Cyclic Natriuretic Peptide Constructs

ABSTRACT

Cyclic constructs with an N-terminus and a C-terminus which bind to a natriuretic peptide receptor and include a plurality of amino acid residues, at least one amino acid surrogate of formula I: 
     
       
         
         
             
             
         
       
     
     where R, R′, Q, Y, W, Z, J, x and n are as defined in the specification, and optionally at least one prosthetic group, pharmaceutical compositions including such cyclic constructs, and methods of treating congestive heart failure or other conditions, syndromes or diseases for which induction of anti-hypertensive, cardiovascular, renal or endocrine effects are desired.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application to U.S. Ser. No.11/694,260 entitled “Cyclic Natriuretic Peptide Constructs”, filed onMar. 30, 2007, now U.S. Pat. No. 7,622,440, issued on Nov. 24, 2009.

U.S. Ser. No. 11/694,260 claims priority to and the benefit of thefiling of U.S. Provisional Patent Application Ser. No. 60/743,960entitled “Cyclic Natriuretic Peptide Constructs”, filed on Mar. 30,2006, and of U.S. Provisional Patent Application Ser. No. 60/743,961entitled “Cyclic Natriuretic Peptide Constructs with Prosthetic Groups”,filed on Mar. 30, 2006, and the specification and claims thereof of eachare incorporated herein by reference.

A related application entitled “Amino Acid Surrogates for PeptidicConstructs” was filed on Mar. 30, 2007, U.S. patent application Ser. No.11/694,181, Attorney Docket No. 0307-043, and the specification andclaims thereof are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention (Technical Field)

The present invention relates to cyclic natriuretic peptide constructswhich include a plurality of amino acid residues, one or morering-constrained amino acid surrogates and optionally one or moreprosthetic groups, which constructs bind a natriuretic peptide receptorand may be employed for therapeutic purposes.

2. Background Art

The natriuretic peptide system has been extensively explored since theidentification of the human atrial natriuretic peptide (ANP) sequenceand gene structure in 1984. ANP is sometimes also called “ANF”, oratrial natriuretic factor. ANP is part of the natriuretic peptidesystem, which in humans involves an ANP gene, which through differencesin post-translational processing results in both ANP and urodilatin, agene which produces BNP, or brain natriuretic peptide, and a gene whichproduces CNP, or c-type natriuretic peptide. ANP, urodilatin, BNP andCNP are each ring structures, with a 17 amino acid loop formed by acysteine-cysteine disulfide linkage. The amino acid sequence andstructure of human ANP (hANP) is shown in FIG. 1. ANP, urodilatin, BNPand CNP are closely related, differing by some five or six amino acidswithin the ring, though the N- and C-terminal tails are substantiallydifferent.

There are three known natriuretic peptide receptors called natriureticpeptide receptors A, B and C (NPRA, NPRB and NPRC). NPRA and NPRB arelinked to guanylyl cyclases, while NPRC is a G-protein linked clearancereceptor. ANP, BNP and CNP are the primary endogenous mammaliannatriuretic peptides identified to date. However, there are a number ofnon-mammalian natriuretic peptides that have been identified and mayhave therapeutic application in mammals. These include salmonnatriuretic or cardiac peptide (sCP), ventricular natriuretic peptide(VNP), a cardiac natriuretic peptide identified in eels and a variety offish, dendroaspis natriuretic peptide (DNP), a natriuretic peptideidentified in mamba snake venom, and three natriuretic-like peptides(TNP-a, TNP-b, and TNP-c) isolated from taipan snake venom. Seegenerally Tervonen V, Ruskoaho H, Lecklin T, Ilves M, Vuolteenaho O,Salmon cardiac natriuretic peptide is a volume-regulating hormone. Am.J. Physiol. Endocrinol. Metab. 283:E353-61 (2002); Takei Y, Fukuzawa A,Itahara Y, Watanabe T X, Yoshizawa Kumagaye K, Nakajima K, Yasuda A,Smith M P, Duff D W, Olson K R. A new natriuretic peptide isolated fromcardiac atria of trout, Oncorhynchus mykiss. FEBS Lett. 414:377-80(1997); Schweitz H, Vigne P, Moinier D, Frelin C, Lazdunski M. A newmember of the natriuretic peptide family is present in the venom of thegreen mamba (Dendroaspis angusticeps). J. Biol. Chem. 267:13928-32(1992); Lisy 0, Jougasaki M, Heublein D M, Schirger J A, Chen H H,Wennberg P W, Burnett J C. Renal actions of synthetic dendroaspisnatriuretic peptide. Kidney Int. 56:502-8 (1999); and Fry B G,Wickramaratana J C, Lemme S, Beuve A, Garbers D, Hodgson W C, Alewood P.Novel natriuretic peptides from the venom of the inland (Oxyuranusmicrolepidotus): isolation, chemical and biological characterisation.Biochem. Biophys. Res. Comm. 327:1011-1015 (2005).

ANP is endogenously secreted predominately in response to increasedatrial pressure, but other factors, including cytokine receptorstimulation, may contribute to endogenous secretion. Once released, ANPis a hormonal regulator of blood pressure, sodium and fluid homeostasis,providing vasorelaxant effects, affecting cardiovascular remodeling, andthe like. Thus ANP, including endogenous ANP, is effective in congestiveheart failure and other cardiovascular disease, in part by providing adefense against a chronically activated renin-angiotensin-aldosteronesystem. Circulating ANP is rapidly removed from the circulation by twomechanisms, binding to a natriuretic peptide receptor and enzymaticdegradation.

Human ANP is also referred to as wild-type human ANP, hANP, ANP(1-28)and ANP(99-126) (the later referring to the relevant sequence withinproANP(1-126), which is normally cleaved at Arg⁹⁸-Ser⁹⁹ in theC-terminal region during secretion). Hereafter human ANP is sometimesreferred to as “hANP.”

In general, natriuretic peptides and variants thereof are believed tohave utility in the treatment of congestive heart failure, renalhypertension, acute kidney failure and related conditions, as well asany condition, disease or syndrome for which a diuretic, natriureticand/or vasodilatory response would have a therapeutic or preventativeeffect. One review article describing natriuretic peptides, includingANP, and use of the natriuretic peptide system in heart failure isSchmitt M., Cockcroft J. R., and Frenneaux M. P. Modulation of thenatriuretic peptide system in heart failure: from bench to bedside?Clinical Science 105:141-160 (2003).

A large number of ANP mimetics and variations have been made, some ofwhich are substantially reduced in size from ANP. On ANP version that isreduced in size yet is biologically active is the 15-mer disulfidecyclic peptideH-Met-cyclo(Cys-His-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Cys)-Tyr-Arg-NH₂(SEQ ID NO:1) as described in Li B, Tom J Y, Oare D, Yen R, FairbrotherW J, Wells J A, Cunningham B C. Minimization of a polypeptide hormone.Science 270:1657-60 (1995). This 15-mer peptide is commonly referred toas “mini-ANP”.

A number of patents and patent applications have been filed on differentsynthetic mimics of natriuretic peptides, asserted to be superior towild-type natriuretic peptides based on one or more factors. Theseinclude the constructs disclosed in the following U.S. Pat. Nos.4,496,544; 4,609,725; 4,656,158; 4,673,732; 4,716,147; 4,757,048;4,764,504; 4,804,650; 4,816,443; 4,824,937; 4,861,755; 4,904,763;4,935,492; 4,952,561; 5,047,397; 5,057,495; 5,057,603; 5,091,366;5,095,004; 5,106,834; 5,114,923; 5,159,061; 5,204,328; 5,212,286;5,352,587; 5,376,635; 5,418,219; 5,665,704; 5,846,932; 5,583,108;5,965,533; 6,028,055; 6,083,982; 6,124,430; 6,150,402; 6,407,211;6,525,022; 6,586,396 and 6,818,619; and in the following U.S. PatentApplication Publications: 2004/0002458; 2004/0063630; 2004/0077537;2005/0113286; 2005/0176641; 2006/0030004. In addition, various non-U.S.patents and patent applications disclose constructs, including: WO85/04870; WO 85/04872; WO 88/03537; WO 88/06596; WO 89/10935; WO89/05654; WO 90/01940; WO 90/14362; WO 92/06998; WO 95/13296; WO99/08510; WO 99/12576; WO 01/016295; WO 2004/047871; WO 2005/072055; EPO0 291 999; EPO 0 323 740; EPO 0 341 603; EPO 0 350 318; EPO 0 356 124;EPO 0 385 476; EPO 0 497 368; and EPO 0 542 863. Chimeric natriureticpeptides, such as a peptide call “vasonatrin peptide” and described as achimera of ANP and CNP, are described, as in U.S. Pat. No. 5,583,108, orin U.S. Pat. Nos. 6,407,211 and 6,818,619, disclosing chimeric peptidesof dendroaspis. The teachings of each of the foregoing patents andpatent applications are incorporated by reference as if set forth infull.

There is one natriuretic peptide product approved by the Food and DrugAdministration in the United States, sold under the generic namenestiritide and the tradename Natrecor® (Scios Inc.). This is a humanB-type natriuretic peptide manufactured from E. coli using recombinantDNA technology. This product is approved only for intravenous infusionfor treatment of patients with actutely decompensated congestive heartfailure who have dyspnea at rest or with minimal activity. Whileeffective, the pharmacokinetics and half-life of nestiritide are suchthat the product can only be employed by intravenous infusion, whichlimits use of the drug to a hospital or skilled medical center setting.

Notwithstanding the large number of compounds that have been developed,virtually none are commercialized or in active clinical development.There is a substantial need for products with improved characteristics,including improved potency, half-life, modes of administration,bioavailability or prolonged duration of effect, which products areeffective for one or more therapeutic indications, and which preferablymay be administered on an out-patient basis.

BRIEF SUMMARY OF THE INVENTION

In one aspect the invention provides a cyclic construct which binds to areceptor for a natriuretic peptide, including but not limited to areceptor for ANP, BNP, CNP, sCP, DNP, TNP-a, TNP-b or TNP-c, whereinsuch construct includes a plurality of amino acid residues, at least oneamino acid surrogate of the general formula I:

where R and R′ are each independently H or a natural or unnatural aminoacid side chain moiety or derivative of an amino acid side chain moiety;x is 1 or 2; Y is CH₂ or C═O; W is CH₂, NH or NR′″; Z is H or CH₃; n is0, 1 or 2; J is —C(═O)— unless the surrogate is at the C-terminusposition of the construct, in which case J is —H, —OH, —C(═O)—OH,—C(═O)—NH₂ or a C-terminus capping group; Q is a bond unless thesurrogate is at the N-terminus position of the construct, in which caseQ is —H or an amine capping group; R′″ is an acyl, a C₁ to C₁₇ linear orbranched alkyl chain, a C₂ to C₁₉ linear or branched alkyl acyl chain, aC₁ to C₁₇ linear or branched omega amino aliphatic, or a C₁ to C₁₇linear or branched omega amino aliphatic acyl; optionally at least oneprosthetic group covalently bonded to a reactive group in a side chainof at least one of the amino acid residues, to an amine capping groupwhere the surrogate is at the N-terminus position of the construct, orto a C-terminus capping group where the surrogate is at the C-terminusposition of the construct; and the carbon atoms marked with an asteriskcan have any stereochemical configuration. The construct is a cyclicconstruct, cyclized by a bond between side chains of two amino acidresidues, between an amino acid residue side chain and an R or R′ groupof an amino acid surrogate, between R or R′ groups of two amino acidsurrogates, between a terminal group of the construct and an amino acidresidue side chain, or between a terminal group of the construct and anR or R′ group of an amino acid surrogate. Preferable the two amino acidresidues forming a bond between the side chains thereof are separated bybetween about eight and ten amino acid residues and optionally zero, oneor two amino acid surrogates. The plurality of amino acid residues mayinclude any amino acid residue selected from the group consisting ofnatural or unnatural α-amino acids, β-amino acids, α, α-disubstitutedamino acids and N-substituted amino acids, including all (R) or (S)configurations of any of the foregoing.

The prosthetic group(s) may include polymeric groups comprising repeatunits including one or more carbon and hydrogen atoms, and optionallyother atoms, including oxygen. Such polymeric groups are preferablywater-soluble polymers, and are preferably poly(alkylene oxide),poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline orpoly(acryloylmorpholine). A preferred poly(alkylene oxide) ispoly(ethylene glycol) (PEG), optionally derivatized with a linkinggroup.

In one aspect, J is a C-terminus capping group selected from

-   —(CH₂)_(m)—OH,-   —C(═O)—(CH₂)_(m)—N(v₁)(v₂),-   —C(═O)—O—(CH₂)_(m)—CH₃,-   —O—(CH₂)_(m)—CH₃,-   —O—(CH₂)_(m)—N(v₁)(v₂),-   —O—(CH₂)_(m)—OH,-   —C(═O)—NH—(CH₂)_(m)—S(v₁),-   —C(═O)—NH—(CH₂)_(m)—CH₃,-   —C(═O)—NH—(CH₂)_(m)—N(v₁)(v₂),-   —C(═O)—N—((CH₂)_(m)—N(v₁)(v₂))₂,-   —C(═O)—NH—CH(—C(═O)—OH)—(CH₂)_(m)—N(v₁)(v₂),-   —C(═O)—NH—(CH₂)_(m)—NH—C(═O)—CH(N(v₁)(v₂))((CH₂)_(m)—N(v₁)(v₂)), or-   —C(═O)—NH—CH(—C(═O)—N(v₁)(v₂))—(CH₂)_(m)—N(v₁)(v₂);    including all (R) or (S) configurations of the foregoing, where v₁    and v₂ are each independently H or a C₁ to C₁₇ linear or branched    alkyl chain and m is in each instance independently 0 to 17.

In another aspect where the amino acid surrogate is at the C-terminusposition of the construct, J is a C-terminus capping group consisting ofan omega amino aliphatic, terminal aryl or aralkyl group or any singlenatural or unnatural α-amino acid, β-amino acid, α, α-disubstitutedamino acid or N-substituted amino acid, including all (R) or (S)configurations of an α, α-disubstituted amino acid where thesubstituents are different, optionally in combination with a C-terminuscapping group as defined above.

In another aspect, Q is an amine capping group selected from

-   —(CH₂)_(m)—N(v₃)(v₄),-   —(CH₂)_(m)—CH₃,-   —(CH₂)_(m)—O(v₃),-   —(CH₂)_(m)—C(═O)-(v₃),-   —(CH₂)_(m)—C(═O)—O-(v₃),-   —(CH₂)_(m)—S(v₃),-   —C(═O)—(CH₂)_(m)—CH₃,-   —C(═O)—(CH₂)_(m)—N(v₃)(v₄),-   —C(═O)—(CH₂)_(m)—C(═O)-(v₃),-   —C(═O)—(CH₂)_(m)—O(v₃), or-   —C(═O)—(CH₂)_(m)—S(v₃);    where v₃ and v₄ are each independently H, a C₁ to C₁₇ linear or    branched alkyl chain or a C₂ to C₁₉ linear or branched alkyl acyl    chain, on the proviso that if one of v₃ or v₄ is an alkyl acyl    chain, then the other of v₃ or v₄ is H, and m is 0 to 17.

In a related aspect, an amino acid surrogate of formula I is at theC-terminus position of the construct, and at least one of R and R′ is anatural or unnatural amino acid side chain moiety or derivative of anamino acid side chain moiety with a heteroatom group comprising at leastone nitrogen atom, and the remaining one of R and R′ is H or a naturalor unnatural amino acid side chain moiety or derivative of an amino acidside chain moiety.

In a related embodiment, the invention provides a construct which bindsto a receptor for a natriuretic peptide, including but not limited to areceptor for ANP, BNP, CNP, sCP, DNP, TNP-a, TNP-b or TNP-c, whereinsuch construct includes a plurality of amino acid residues and at leastone amino acid surrogate located at any position other than theC-terminus position or N-terminus position and covalently bonded by twopeptide bonds, and of formula II:

where R and R′ are each independently H or a natural or unnatural aminoacid side chain moiety or derivative of an amino acid side chain moiety;x is 1 or 2; Y is CH₂ or C═O; W is CH₂, NH or NR′″; Z is H or CH₃; R′″is an acyl, a C₁ to C₁₇ linear or branched alkyl chain, a C₂ to C₁₉linear or branched alkyl acyl chain, a C₁ to C₁₇ linear or branchedomega amino aliphatic, or a C₁ to C₁₇ linear or branched omega aminoaliphatic acyl; n is 0, 1 or 2; the carbon atoms marked with an asteriskcan have any stereochemical configuration; and the broken lines indicatethe bond forming a peptide bond.

Where the surrogate of formula I is at the C-terminus of the construct,it is covalently bonded thereto by a single peptide bond, such that thesurrogate has the formula:

where the broken line indicates the bond forming a peptide bond. Wherethe surrogate is at the N-terminus of the construct it is preferably offormula I, and is covalently bonded thereto by a single bond peptidebond, such that the surrogate has the formula:

where the broken line indicates the bond forming a peptide bond.However, where the surrogate is at other than at the N-terminus orC-terminus of the construct, it is preferably of formula II and iscovalently bonded thereto by two peptide bonds.

In different embodiments of the invention, one amino acid surrogate maybe employed in a construct of the invention, two amino acid surrogatesmay be employed in a construct of the invention, or more than two aminoacid surrogates may be employed in a construct of the invention.

In another preferred embodiment, the invention provides a constructwherein one or more peptide bonds between amino acid residues aresubstituted with a non-peptide bond.

A primary object of the present invention is to provide natriureticreceptor-specific constructs.

Another object of the present invention is to provide natriureticreceptor-specific constructs wherein one or more amino acid residues aresubstituted by a ring-constrained amino acid surrogate.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the construct exhibits, uponadministration to a mammal, one or more advantages relative to thecorresponding amino acid sequence not comprising an amino acidsurrogate, the advantages selected from the group consisting ofincreased resistance to enzymatic degradation, increased circulationhalf life, increased bioavailability, increased efficacy, prolongedduration of effect and combinations of the foregoing.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the construct has at least 10% ofthe maximal cGMP stimulating activity as the same concentration of thecorresponding amino acid sequence not comprising an amino acidsurrogate.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the construct has at least 50% ofthe maximal cGMP stimulating activity as the same concentration of thecorresponding amino acid sequence not comprising an amino acidsurrogate.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the construct has at least 100% ofthe maximal cGMP stimulating activity as the same concentration of thecorresponding amino acid sequence not comprising an amino acidsurrogate.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the construct has more than 100% ofthe maximal cGMP stimulating activity as the same concentration of thecorresponding amino acid sequence not comprising an amino acidsurrogate.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the construct has an equilibriumreceptor binding affinity, determined by the Ki (nM) value, no greaterthan two log orders higher than the Ki (nM) value of the correspondingamino acid sequence not comprising an amino acid surrogate.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the construct has an equilibriumreceptor binding affinity, determined by the Ki (nM) value, no greaterthan three times higher than the Ki (nM) value of the correspondingamino acid sequence not comprising an amino acid surrogate.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the construct has an equilibriumreceptor binding affinity, determined by the Ki (nM) value, equal to orless than than the Ki (nM) value of the corresponding amino acidsequence not comprising an amino acid surrogate.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the construct has an equilibriumreceptor binding affinity, determined by the Ki (nM) value, less thanthe Ki (nM) value of the corresponding amino acid sequence notcomprising an amino acid surrogate.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the construct has a receptor bindingaffinity with respect to a natriuretic peptide receptor greater than thereceptor binding affinity of the corresponding amino acid sequence notcomprising an amino acid surrogate.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the construct has biologicalefficacy, determined by decrease in blood pressure or increase in urineoutput over time, at least as efficacious as or more efficacious thanthan the same dose of the corresponding amino acid sequence notcomprising an amino acid surrogate.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the construct has biologicalefficacy, determined by decrease in blood pressure or increase in urineoutput over time, more efficacious than than the same dose of thecorresponding amino acid sequence not comprising an amino acidsurrogate.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the corresponding amino acidsequence not comprising an amino acid surrogate has at least about 60%homology with the sequence of a natriuretic peptide.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the corresponding amino acidsequence not comprising an amino acid surrogate has at least about 80%homology with the sequence of a natriuretic peptide.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the corresponding amino acidsequence not comprising an amino acid surrogate has at least about 60%homology with the sequence of a peptide that binds to a receptor forANP, BNP, CNP, sCP, DNP, TNP-a, TNP-b or TNP-c.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the corresponding amino acidsequence not comprising an amino acid surrogate has at least about 80%homology with the sequence of a peptide that binds to a receptor forANP, BNP, CNP, sCP, DNP, TNP-a, TNP-b or TNP-c.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the corresponding amino acidsequence not comprising an amino acid surrogate has at least about 60%homology with the sequenceH-Met-cyclo(Cys-His-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Cys)-Tyr-Arg-NH₂(SEQ ID NO:1).

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the corresponding amino acidsequence not comprising an amino acid surrogate has at least about 80%homology with the sequenceH-Met-cyclo(Cys-His-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Cys)-Tyr-Arg-NH₂(SEQ ID NO:1).

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the corresponding amino acidsequence not comprising an amino acid surrogate has at least about 60%homology with the sequenceH-Met-cyclo(Xaa-His-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Xaa)-Tyr-Arg-NH₂(SEQ ID NO:2), where Xaa are each independently any amino acid residuetogether forming a cyclic peptide.

Another object of the present invention is to provide a natriureticreceptor-specific construct wherein the corresponding amino acidsequence not comprising an amino acid surrogate has at least about 80%homology with the sequenceH-Met-cyclo(Xaa-His-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Xaa)-Tyr-Arg-NH₂(SEQ ID NO:2), where Xaa are each independently any amino acid residuetogether forming a cyclic peptide.

Another object of the present invention is to provide a natriureticreceptor-specific construct including a surrogate as defined hereinwherein the corresponding amino acid sequence not comprising an aminoacid surrogate is a peptide which binds to a receptor for ANP.

Another object of the present invention is to provide a natriureticreceptor-specific construct including a surrogate as defined hereinwherein the corresponding amino acid sequence not comprising an aminoacid surrogate is a peptide which binds to a receptor for BNP.

Another object of the present invention is to provide natriureticreceptor-specific constructs with greater bioavailability and half-lifethan natural or recombinant forms of ANP or BNP.

Another object of the present invention is to provide natriureticreceptor-specific constructs which may be administered to patients withcongestive heart failure.

Another object of the present invention is to provide natriureticreceptor-specific constructs which may be administered by at least oneroute of administration in addition to intravenous administration.

Another object of the present invention is to provide natriureticreceptor-specific constructs which may be administered to patients bysubcutaneous or intramuscular injection.

Another object of the present invention is to provide natriureticreceptor-specific constructs with increased resistance to degradationbut which have a significantly high binding affinity to its receptor.

Another object of the present invention is to provide natriureticreceptor-specific constructs in a sustained release formulation.

Other objects, advantages and novel features, and further scope ofapplicability of the present invention will be set forth in part in thedetailed description to follow, taken in conjunction with theaccompanying drawings, and in part will become apparent to those skilledin the art upon examination of the following, or may be learned bypractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the instrumentalities andcombinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of thepresent invention and, together with the description, serve to explainprinciples of the invention. The drawings are only for the purpose ofillustrating one or more preferred embodiments of the invention and arenot to be construed as limiting the invention. In the drawings:

FIG. 1 is the sequence of wild-type endogenous human ANP (hANP);

FIG. 2 is a graph of the concentration of construct 1-18 in rats overtime when administered by subcutaneous means at 5 mg/kg and byintravenous means at 2 mg/kg;

FIG. 3 is a graph of total urine output over thirty minutes in a groupof four rats when administered constructs 1-18 and 1-63 by IV routes;and,

FIG. 4 is a graph of total urine output over forty-five minutes in agroup of four rats when administered construct 1-18 in different dosesby SC routes.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides natriuretic receptor-specific constructs made ofa plurality of amino acid residues, at least one ring-constrained aminoacid surrogate and optionally at least one prosthetic group. Thering-constrained amino acid surrogates employed in the invention arepreferably such that they may be made with a conventional aminoprotected N-terminus, using a protecting group such as Fmoc, and areactive carboxyl C-terminus, and may thus be employed in conventionalpeptide synthesis methodologies, it being understood that if the aminoacid surrogate is at the C-terminus position of the construct, thatother than a carboxyl terminus may be employed on such surrogate. Thus,in a preferred embodiment the invention provides synthetically madeconstructs, synthesized using peptide synthesis methodologies modifiedas appropriate, and comprising a plurality of amino acid residues and atleast one ring-constrained amino acid surrogate, In a related preferredembodiment, the construct further includes at least one prostheticgroup.

Preferred prosthetic groups include polymeric groups comprising repeatunits including one or more carbon and hydrogen atoms, and optionallyother atoms, including oxygen. Such polymeric groups are preferablywater-soluble polymers, and are preferably poly(alkylene oxide),poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline orpoly(acryloylmorpholine). A preferred poly(alkylene oxide) ispoly(ethylene glycol) (PEG), optionally derivatized with a linkinggroup.

In one aspect, the invention provides a construct with an amino acidsequence that is a homolog of a known natriuretic peptide, such as ANPor BNP, or is a homolog of any known peptide variant of a natriureticpeptide, wherein the construct includes at least one amino acidsurrogate of formula I or II. The corresponding amino acid sequence notcomprising an amino acid surrogate may be identical to a knownnatriuretic peptide or a known peptide variant, or may be homologousthereto, such as a corresponding amino acid sequence that is at least60% homologous, or more preferably is at least about 80% homologous. Asused herein, the phrase “corresponding amino acid sequence notcomprising an amino acid surrogate” means an amino acid sequence,including a known amino acid sequence, that binds to a receptor for anatriuretic peptide and that does not include a surrogate. Such knownamino acid sequence is identical to the construct if the amino acidsequence is the same but for the substitution by or addition of one ormore amino acid surrogates. Similarly, homology is determined byreference to identity of the known amino acid sequence to the constructbut for the substitution by or addition of one or more amino acidsurrogates.

In another aspect, the invention provides a construct that is modeled ona known peptide which binds to a receptor for a natriuretic peptide, butwhich includes one or more amino acid surrogates, such surrogates beingeither substituted for one or more amino acid residues contained in theknown peptide, or in addition to the sequence comprising the knownpeptide. The known peptide may be any natriuretic peptide known in theart, including but not limited to those disclosed in any publication,patent, application or reference cited herein, including but not limitedto the natriuretic peptides disclosed in U.S. Pat. Nos. 4,496,544;4,609,725; 4,656,158; 4,673,732; 4,716,147; 4,757,048; 4,764,504;4,804,650; 4,816,443; 4,824,937; 4,861,755; 4,904,763; 4,935,492;4,952,561; 5,047,397; 5,057,495; 5,057,603; 5,091,366; 5,095,004;5,106,834; 5,114,923; 5,159,061; 5,204,328; 5,212,286; 5,352,587;5,376,635; 5,418,219; 5,665,704; 5,846,932; 5,583,108; 5,965,533;6,028,055; 6,083,982; 6,124,430; 6,150,402; 6,407,211; 6,525,022;6,586,396 or 6,818,619; in U.S. Patent Application Publications2004/0002458; 2004/0063630; 2004/0077537; 2005/0113286; 2005/0176641; or2006/0030004; or in various non-U.S. patents and patent applications,including WO 85/04870; WO 85/04872; WO 88/03537; WO 88/06596; WO89/10935; WO 89/05654; WO 90/01940; WO 90/14362; WO 92/06998; WO95/13296; WO 99/08510; WO 99/12576; WO 01/016295; WO 2004/047871; WO2005/072055; EPO 0 291 999; EPO 0 323 740; EPO 0 341 603; EPO 0 350 318;EPO 0 356 124; EPO 0 385 476; EPO 0 497 368; or EPO 0 542 863. In oneaspect, the known peptide is a peptide or homolog thereof disclosed inU.S. Pat. Nos. 4,656,158, 4,824,937, 4,935,492, 5,159,061, 5,204,328,5,376,635, 5,665,704, 5,846,932, 6,028,055, 6,407,211, 6,525,022,6,586,396, or 6,818,619, U.S. Patent Application Publications2004/0002458, 2004/0063630, or 2005/0176641, or International PatentApplication Publications WO 2004/047871 or WO 2005/072055. The teachingsof each of the foregoing patents and patent applications areincorporated by reference as if set forth in full.

In one particularly preferred embodiment, the invention provides aconstruct, comprising an amino acid sequence which binds to anatriuretic peptide receptor, wherein one or more amino acid residues insuch amino acid sequence which binds to a natriuretic peptide receptoris substituted with an amino acid surrogate of formula I. In one aspect,the amino acid sequence which binds to a natriuretic peptide receptoris, prior to substitution,H-Met-cyclo(Cys-His-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Cys)-Tyr-Arg-NH₂(SEQ ID NO:1).

In yet another aspect the invention provides a construct that binds to areceptor for a natriuretic peptide, including a receptor for ANP or BNP,and includes at least one amino acid surrogate of formula I or II, butwhich construct is not homologous to any known peptide that binds to areceptor for a natriuretic peptide.

In one embodiment, the invention provides a cyclic construct of formulaIII:

where

Aaa¹ is an L- or D-isomer of an α-amino acid or β-amino acid or an α,α-disubstituted amino acid derived from an α-amino acid, including whereAaa¹ is an L- or D-isomer of an α-amino acid or β-amino acid includingor derived from Nle, Ala, Leu, Ile, Val, Arg, Phe, Lys, Tyr, Asp, Nva,Met, Met(O), or Met(O₂), or an α, α-disubstituted amino acid derivedfrom Nle, Ala, Leu, Ile, Val, Arg, Phe, Lys, Tyr, Asp, Nva, Met, Met(O),or Met(O₂), including all (R) or (S) configurations of α,α-disubstituted amino acids where the substituents are different, orAaa¹ is an acyl comprising a C₂ to C₁₈ linear alkyl, a C₃ to C₁₇branched alkyl, a C₂ to C₁₈ linear alkenyl or alkynyl or a C₃ to C₁₈branched alkenyl or alkynyl, or Aaa¹ is an amino acid surrogate of thestructure:

wherein the broken line indicates a peptide bond; R and R′ areindependently H, a linear or branched C₁ to C₆ aliphatic chain,—(CH₂)_(y)—S—CH₃, —(CH₂)_(y)—S(═O)—CH₃, —(CH₂)_(y)—S(O₂)—CH₃, a bond anda cyclopropane, cyclobutane, cyclopentane, or cyclohexane ring, or a C₁to C₃ aliphatic chain and a cyclopropane, cyclobutane, cyclopentane, orcyclohexane ring; x is 1 or 2; Y is CH₂ or C═O; W is CH₂, NH or NR′″; Zis H or CH₃; Q is —H, —(CH₂)_(m)—N(v₃)(v₄), —(CH₂)_(m)—CH₃,—(CH₂)_(m)—O(v₃), —(CH₂)_(m)—C(═O)-(v₃), —(CH₂)_(m)—C(═O)—O-(v₃),—(CH₂)_(m)—S(v₃), —C(═O)—(CH₂)_(m)—CH₃, —C(═O)—(CH₂)_(m)—N(v₃)(v₄),—C(═O)—(CH₂)_(m)—C(═O)-(v₃), —C(═O)—(CH₂)_(m)—O(v₃), or—C(═O)—(CH₂)_(m)—S(v₃); R′″ is an acyl, a C₁ to C₁₇ linear or branchedalkyl chain, a C₂ to C₁₉ linear or branched alkyl acyl chain, a C₁ toC₁₇ linear or branched omega amino aliphatic, or a C₁ to C₁₇ linear orbranched omega amino aliphatic acyl; n is 0, 1 or 2; m is 0 to 17; y is1 to 5; v₃ and v₄ are each independently H, a C₁ to C₁₇ linear orbranched alkyl chain or a C₂ to C₁₉ linear or branched alkyl acyl chain,on the proviso that if one of v₃ or v₄ is an alkyl acyl chain, then theother of v₃ or v₄ is H; and the carbon atoms marked with an asterisk canhave any stereochemical configuration;

Aaa² and Aaa¹³ are the same or different, and are each L- or D-isomeramino acid residues forming a cyclic bridge through the side chains ofeach of Aaa² and Aaa¹³ wherein the linking group of the cyclic bridge is—S—S—, —S—CH₂—S—, —S—CH₂—, —CH₂—S—, —C(═O)—NH—, —NH—C(═O)—, —CH₂—NH—,—NH—CH₂—, —CH₂—S(O)_(n)— where n is 1 or 2, —S(O)_(n)—CH₂— where n is 1or 2, —CH₂—CH₂—, —CH═CH— (E or Z), —C≡C—, —C(═O)—O—, —O—C(═O)—,—C(═O)—CH₂—, —CH₂—C(═O)—, —O—C(═O)—NH—, —NH—C(═O)—O—, or —NH—C(═O)—NH—;

Aaa³ is an L- or D-isomer of an α-amino acid or β-amino acid includingor derived from His, Ala, Ser, Thr, Lys, HLys, Orn, Cys, HCys, Dap, orDab, or an α, α-disubstituted amino acid derived from His, Ala, Ser,Thr, Lys, HLys, Orn, Cys, HCys, Dap, or Dab, including all (R) or (S)configurations of α, α-disubstituted amino acids where the substituentsare different, or Aaa³ is an amino acid surrogate of the structure:

where R and R′ are independently H or an amino acid side chain moiety ofHis, Ala, Ser, Thr, Lys, HLys, Orn, Cys, HCys, Dap, or Dab or aderivative of an amino acid side chain moiety of His, Ala, Ser, Thr,Lys, HLys, Orn, Cys, HCys, Dap, or Dab; x is 1 or 2; Y is CH₂ or C═O; Wis CH₂, NH or NR′″; Z is H or CH₃; R′″ is an acyl, a C₁ to C₁₇ linear orbranched alkyl chain, a C₂ to C₁₉ linear or branched alkyl acyl chain, aC₁ to C₁₇ linear or branched omega amino aliphatic, or a C₁ to C₁₇linear or branched omega amino aliphatic acyl; and n is 0, 1 or 2;

Aaa⁴ is an L- or D-isomer of an α-amino acid or β-amino acid includingor derived from substituted or unsubstitued Phe, HPhe or Pgl, or Tyr,Leu, Ile, Val, Ala, Nle, Nva or Tle, or an α, α-disubstituted amino acidderived from substituted or unsubstitued Phe, HPhe or Pgl, or Tyr, Leu,Ile, Val, Ala, Nle, Nva or Tle, including all (R) or (S) configurationsof α, α-disubstituted amino acids where the substituents are different,or Aaa⁴ is an amino acid surrogate as for Aaa³ where R and R′ areindependently H or an amino acid side chain moiety of substituted orunsubstitued Phe, HPhe or Pgl, or Tyr, Leu, Ile, Val, Ala, Nle, Nva orTle or a derivative of an amino acid side chain moiety of substituted orunsubstitued Phe, HPhe or Pgl, or Tyr, Leu, Ile, Val, Ala, Nle, Nva orTle;

Aaa⁵ is Gly, Sar, an L- or D-isomer of an α-amino acid or β-amino acidincluding or derived from Ala, or Aib, which is the α, α-disubstitutedamino acid derived from Ala, or Aaa⁵ is an amino acid surrogate as forAaa³ where R and R′ are independently H or —CH₃;

Aaa⁶ is Gly, Sar, an L- or D-isomer of an α-amino acid or β-amino acidincluding or derived from Ala, or Aib, or Aaa⁶ is an amino acidsurrogate as for Aaa³ where R and R′ are independently H or —CH₃;

Aaa⁷ is an L- or D-isomer of an α-amino acid or β-amino acid includingor derived from Arg, His, Ala, Ser, HSer, Thr, Lys, HLys, Orn, Cys,HCys, Cit, Abu, Dap, or Dab, or an α, α-disubstituted amino acid derivedfrom Arg, His, Ala, Ser, HSer, Thr, Lys, HLys, Orn, Cys, HCys, Cit, Abu,Dap, or Dab, including all (R) or (S) configurations of α,α-disubstituted amino acids where the substituents are different, orAaa⁷ is an amino acid surrogate as for Aaa³ where R and R′ areindependently H or an amino acid side chain moiety of Arg, His, Ala,Ser, HSer, Thr, Lys, HLys, Orn, Cys, HCys, Abu, Dap, or Dab or aderivative of an amino acid side chain moiety of Arg, His, Ala, Ser,HSer, Thr, Lys, HLys, Orn, Cys, HCys, Abu, Dap, or Dab;

Aaa⁸ is Gly, an L- or D-isomer of an α-amino acid or β-amino acidincluding or derived from Nle, Ile, Leu, Val, Phe, Ala, Nva, Met(O),Met(O₂), or Tle, or an α, α-disubstituted amino acid derived from Nle,Ile, Leu, Val, Phe, Ala, Nva, Met(O), Met(O₂), or Tle, including all (R)or (S) configurations of α, α-disubstituted amino acids where thesubstituents are different, or Aaa⁸ is an amino acid surrogate as forAaa³ where R and R′ are independently H or an amino acid side chainmoiety of Nle, Ile, Leu, Val, Phe, Ala, Nva, Met(O), Met(O₂), or Tle, ora derivative of an amino acid side chain moiety of Nle, Ile, Leu, Val,Phe, Ala, Nva, Met(O), Met(O₂), or Tle;

Aaa⁹ is an L- or D-isomer of an α-amino acid or β-amino acid includingor derived from Asp, Glu, His, Ala, Ser, Thr, Lys, HLys, Cys, HCys,Met(O), Met(O₂), Orn, Dap, or Dab, or an α, α-disubstituted amino acidderived from Asp, Glu, His, Ala, Ser, Thr, Lys, HLys, Cys, HCys, Met(O),Met(O₂), Orn, Dap, or Dab, including all (R) or (S) configurations of α,α-disubstituted amino acids where the substituents are different, orAaa⁹ is an amino acid surrogate as for Aaa³ where R and R′ areindependently H or an amino acid side chain moiety of Asp, Glu, His,Ala, Ser, Thr, Lys, HLys, Cys, HCys, Met(O), Met(O₂), Orn, Dap, or Dabor a derivative of an amino acid side chain moiety of Asp, Glu, His,Ala, Ser, Thr, Lys, HLys, Cys, HCys, Met(O), Met(O₂), Orn, Dap, or Dab;

Aaa¹⁰ is an L- or D-isomer of an α-amino acid or β-amino acid includingor derived from Arg, His, Ala, Ser, Thr, Lys, HLys, Cys, HCys, Cit,Met(O), Orn, Dap, or Dab, or an α, α-disubstituted amino acid derivedfrom Arg, His, Ala, Ser, Thr, Lys, HLys, Cys, HCys, Cit, Met(O), Orn,Dap, or Dab, including all (R) or (S) configurations of α,α-disubstituted amino acids where the substituents are different, orAaa¹⁰ is an amino acid surrogate as for Aaa³ where R and R′ areindependently H or an amino acid side chain moiety of Arg, His, Ala,Ser, Thr, Lys, HLys, Cys, HCys, Met(O), Orn, Dap, or Dab or a derivativeof an amino acid side chain moiety of Arg, His, Ala, Ser, Thr, Lys,HLys, Cys, HCys, Met(O), Orn, Dap, or Dab;

Aaa¹¹ is Gly or a D- or L-isomer of an α-amino acid or β-amino acidincluding or derived from Nle, Ile, Leu, Val, Phe, Ala, Nva, Cys, HCys,Abu or Tle, or an α, α-disubstituted amino acid derived from Nle, Ile,Leu, Val, Phe, Ala, Nva, Cys, HCys, Abu or Tle, including all (R) or (S)configurations of α, α-disubstituted amino acids where the substituentsare different, or Aaa¹¹ is an amino acid surrogate as for Aaa³ where Rand R′ are independently H or an amino acid side chain moiety of Nle,Ile, Leu, Val, Phe, Ala, Nva, Cys, HCys, Abu or Tle or a derivative ofan amino acid side chain moiety of Nle, Ile, Leu, Val, Phe, Ala, Nva,Cys, HCys, Abu or Tle;

Aaa¹² is Gly, an L- or D-isomer of an α-amino acid or β-amino acidincluding or derived from Ser, Nle, Ile, Leu, Val, Phe, Ala, Nva, Arg,Lys, Orn, Cys, HCys, Abu or Tle, or an α, α-disubstituted amino acidderived from Ser, Nle, Ile, Leu, Val, Phe, Ala, Nva, Arg, Lys, Orn, Cys,HCys, Abu or Tle, including all (R) or (S) configurations of α,α-disubstituted amino acids where the substituents are different, orAaa¹² is an amino acid surrogate as for Aaa³ where R and R′ areindependently H or an amino acid side chain moiety of Ser, Nle, Ile,Leu, Val, Phe, Ala, Nva, Arg, Lys, Orn, Cys, HCys, Abu or Tle or aderivative of an amino acid side chain moiety of Ser, Nle, Ile, Leu,Val, Phe, Ala, Nva, Arg, Lys, Orn, Cys, HCys, Abu or Tle;

Aaa¹⁴ is an L- or D-isomer of an α-amino acid or β-amino acid includingor derived from substituted or unsubstitued Phe, HPhe or Pgl, or Tyr,Leu, Ile, Val, Ala, Lys, Orn, Nle, Nva or Tle, or an α, α-disubstitutedamino acid derived from substituted or unsubstitued Phe, HPhe or Pgl, orTyr, Leu, Ile, Val, Ala, Lys, Orn, Nle, Nva or Tle, including all (R) or(S) configurations of α, α-disubstituted amino acids where thesubstituents are different, or Aaa¹⁴ is an amino acid surrogate of thestructure of formula II as for Aaa³ where R and R′ are independently Hor an amino acid side chain moiety of substituted or unsubstitued Phe,HPhe or Pgl, or Tyr, Leu, Ile, Val, Ala, Lys, Orn, Nle, Nva or Tle or aderivative of an amino acid side chain moiety of substituted orunsubstitued Phe, HPhe or Pgl, or Tyr, Leu, Ile, Val, Ala, Lys, Orn,Nle, Nva or Tle; and

Aaa¹⁵ is a D- or L-isomer of an α-amino acid or β-amino acid includingor derived from Ala, Arg, Orn, Lys, Ala, Dap, Dab, HArg, or HLys, or anα, α-disubstituted amino acid derived from Ala, Arg, Orn, Lys, Ala, Dap,Dab, HArg, or HLys, including all (R) or (S) configurations of α,α-disubstituted amino acids where the substituents are different, orAaa¹⁵ is an amino acid surrogate of the structure:

wherein the broken line indicates a peptide bond; at least one of R andR′ is —(CH₂)_(y)—R″ and if one, the remaining of R and R′ is H, where R″is:

-   —NH₂,-   —NH—C(═NH)—NH₂,-   —NH—(CH₂)_(y)—NH₂,-   —NH—C(═O)—NH₂,-   —C(═O)—NH₂,-   —C(═O)—NH—CH₃,-   —C(═O)—NH—(CH₂)_(y)—NH₂,-   —NH—C(═NH)—NH-Me,-   —NH—C(═NH)—NH-Et,-   —NH—C(═NH)—NH—Pr,-   —NH—C(═NH)—NH—Pr-i,-   —NH—C(═O)—CH₃,-   —NH—C(═O)—CH₂—CH₃,-   —NH—C(═O)—CH—(CH₃)₂,-   —NH—C(═O)—O—CH₃,-   —NH—C(═O)—O—CH₂—CH₃,-   —NH—C(═O)—O—C—(CH₃)₃,-   —NH—C(═O)—NH—CH₃,-   —NH—C(═N—C(═O)—O—C—(CH₃)₃)—NH—C(═O)—O—C—(CH₃)₃,-   —N(C(═O)—O—C—(CH₃)₃)—C(═NH)—NH—C(═O)—O—C—(CH₃)₃,

x is 1 or 2; Y is CH₂ or C═O; W is CH₂, NH or NR′″; Z is H or CH₃; J is—H, —(CH₂)_(m)—OH, —C(═O)—CH₂)_(m)—OH, —C(═O)—CH₂)_(m)—N(v₁)(v₂),—C(═O)—O—(CH₂)_(m)—CH₃, —O—(CH₂)_(m)—CH₃, —O—(CH₂)_(m)—N(v₁)(v₂),—O—(CH₂)_(m)—OH, —C(═O)—NH—(CH₂)_(m)—CH₃, —C(═O)—NH—(CH₂)_(m)—N(v₁)(v₂),—C(═O)—NH—(CH₂)_(m)—S(v₁), —C(═O)—N—((CH₂)_(m)—N(v₁)(v₂))₂,—C(═O)—NH—CH(—C(═O)—OH)—(CH₂)_(m)—N(v₁)(v₂),—C(═O)—NH—(CH₂)_(m)—NH—C(═O)—CH(N(v₁)(v₂))((CH₂)_(m)—N(v₁)(v₂)),—C(═O)—NH—CH(—C(═O)—N(v₁)(v₂))—(CH₂)_(m)—N(v₁)(v₂), an omega aminoaliphatic, terminal aryl or aralkyl group, any single natural orunnatural α-amino acid, β-amino acid or α, α-disubstituted amino acid incombination with one of the foregoing groups defining J, or any singlenatural or unnatural α-amino acid, β-amino acid or α, α-disubstitutedamino acid, including all (R) and (S) configurations of any of theforegoing; R′″ is an acyl, a C₁ to C₁₇ linear or branched alkyl chain, aC₂ to C₁₉ linear or branched alkyl acyl chain, a C₁ to C₁₇ linear orbranched omega amino aliphatic, or a C₁ to C₁₇ linear or branched omegaamino aliphatic acyl; v₁ and v₂ are each independently H or a C₁ to C₁₇linear or branched alkyl chain; n is 0, 1 or 2; m is 0 to 17; y is 1 to5; and the carbon atoms marked with an asterisk can have anystereochemical configuration;

on the proviso that at least one of Aaa¹, Aaa³ through Aaa¹², Aaa¹⁴ orAaa¹⁵ is an amino acid surrogate.

A related embodiment of formula III provides a construct where one ormore of Aaa¹, Aaa³ to Aaa¹², Aaa¹⁴ or Aaa¹⁵ is an amino acid surrogateas defined above, and where a prosthetic group, as hereafter defined, isattached to a reactive group of a side chain of an amino acid residue atone or more of Aaa¹, Aaa³ to Aaa¹², Aaa¹⁴ or Aaa¹⁵, to a reactive R orR′ group of an amino acid surrogate at Aaa³ to Aaa¹² or Aaa¹⁴, directlyor through a Q group to the terminal amine of an amino acid surrogate atAaa¹, to a reactive terminal carboxyl of an amino acid surrogate atAaa⁵, or to a reactive group forming a part of J of an amino acidsurrogate at Aaa¹⁵. The reactive group to which the one or moreprosthetic groups are covalently bonded may be a primary amine, asecondary amine, a carboxyl group, a thiol group or a hydroxyl group. Inone aspect, the prosthetic group may be covalently bound to a reactiveamine in position Aaa¹, Aaa³, Aaa⁷, Aaa¹⁰, Aaa¹² or Aaa¹⁵ or acombination of the foregoing. In another aspect, the prosthetic groupmay be covalently bound to a reactive carboxyl in position Aaa⁹ or Aaa¹⁵or both. In another aspect, the prosthetic group may be covalently boundto a reactive thiol in position Aaa³, Aaa⁷, Aaa⁹, Aaa¹⁰, Aaa¹¹, or Aaa¹²or a combination of the foregoing.

In a preferred aspect of the construct of formula III, one, two or threeof Aaa¹ to Aaa¹⁵ (excluding Aaa² and Aaa¹³) are an amino acid surrogateof one of the foregoing formulas. In a first particularly preferredaspect, one of Aaa¹, Aaa⁵ and Aaa¹⁵ is an amino acid surrogate. In asecond particularly preferred aspect, two of Aaa¹, Aaa⁵ and Aaa¹⁵ areamino acid surrogates. In a third particularly preferred aspect, each ofAaa¹, Aaa⁵ and Aaa¹⁵ are amino acid surrogates. In another particularlypreferred aspect, one, two or three of Aaa¹, Aaa⁵ and Aaa¹⁵ are aminoacid surrogates, and the construct is a cyclic construct formed bydisulfide bond formation through the side chains of Aaa² and Aaa¹³. Inanother particularly preferred aspect, where two or more of Aaa¹ toAaa¹⁵ are amino acid surrogates the amino acid surrogates are notcontiguous, which is to say that each amino acid surrogate is separatefrom each other amino acid surrogate by at least one amino acid residuebeing interposed therebetween in the primary sequence.

In yet another preferred embodiment, in the construct of formula III atleast one of Aaa³, Aaa⁵, Aaa⁶, Aaa⁷, Aaa⁹, Aaa¹⁰, or Aaa¹² is an L- orD-isomer of Ala, preferably an L-isomer of Ala.

In yet another embodiment, the invention provides a construct of formulaIII further comprising one or more non-peptide bonds. Non-peptide bondsmay be employed to decrease the susceptibility of a construct of theinvention to degradation, such as improving the in vivo stability ofconstructs towards tryptic-like proteases by replacing the nativepeptide bond before each Lys or Arg residue with a non-peptide bond,such as an isostere of an amide, a substituted amide or a peptidomimeticlinkage. In one specific embodiment, native peptide bonds are replacedwith peptide bonds having a reversed polarity. In general, anynon-peptide bond may be employed, and may be utilized between any tworesidues. A non-peptide bond includes bonds in which the carbon atomparticipating in the bond between two residues is reduced from acarbonyl carbon to a methylene carbon, such as a non-peptide bond—CH₂—NH— or its isostere —NH—CH₂—, or the use of other bonds such as—CH₂—S—, —CH₂—O—, or —C(═O)—CH₂— or an isostere of any of the foregoing,or —CH₂—CH₂— or —CH═CH—. In general, non-peptide bonds include an imino,ester, hydrazine, semicarbazide, oxime, or azo bond.

The constructs defined above may include one or more prosthetic groups.Prosthetic groups may be employed to modulate the residence time incirculation, to modulate bioavailability, modulate immunogenicity ofconstructs, or the like. In general, prosthetic groups “modulate” byincreasing the residence time, bioavailability or the like, as the casemay be, but prosthetic groups may optionally decrease residence time,bioavailability or the like. A “prosthetic group” thus includes anycompound conjugated, such as by a covalent bond, to a construct of anyformula, for purposes of improving pharmacokinetic or pharmacodynamicproperties of the construct. Preferred prosthetic groups includepolymeric groups, comprising repeat units which in turn comprise one ormore carbon and hydrogen atoms, and optionally other atoms, includingoxygen atoms. Such polymeric groups are preferably water-solublepolymers, and are preferably poly(alkylene oxide), poly(vinylpyrrolidone), poly(vinyl alcohol), polyoxazoline orpoly(acryloylmorpholine). A preferred poly(alkylene oxide) ispoly(ethylene glycol) (PEG). In addition to PEG, other poly(alkyleneglycol) polymers may be employed, such as poly(propylene glycol) andpoly(butylene glycol).

In one embodiment, the prosthetic group is one or more PEG polymerscovalently bound to a reactive group of the construct. The PEG polymer,or other prosthetic group, may be covalently bound to a reactive groupon the side chain of one or more amino acid residues, or may becovalently bound to a reactive group on an amino acid surrogate. Suchreactive groups of an amino acid surrogate may include a groupcovalently bound, directly or through one or more intermediates, to Q orJ, or may include a reactive group forming a part of R or R′.

If PEG is employed as the prosthetic group, the PEG polymer may have amolecular weight of from about 200 MW to about 50000 MW. The PEG polymermay be linear, and if linear, may be monofunctional, with a reactivegroup at one end and a non-reactive group at the other end,homobifunctional, with the same reactive group at each end, orheterobifunctional, with a different reactive group at each end.Alternatively, the PEG polymer may be branched, having generally a“Y”-shaped configuration, multi-armed, such as with two, three, four oreight arms, or other configurations known in the art. The PEG polymerpreferably has at least one derivatized reactive group for linking toone or more defined groups on the construct of any of formula IIIthrough XIII, preferably by means of a covalent bond. The derivativizedreactive group may link to, for example, an amine, hydroxyl, thiol, orcarboxyl group on a construct, including on a terminal group of an aminoacid residue, on a side chain of an amino acid residue, on a Q group ofa surrogate, on a J group of a surrogate, or on an R or R′ group of asurrogate.

The PEG polymer preferably has, at one end, an end-cap group, such as ahydroxyl, alkoxy, substituted alkoxy, aleknoxy, substituted alkenoxy,alkynoxy, substituted alkynoxy, aryloxy or substituted aryloxy. The PEGpolymer further preferably has, at least one other end, a derivatizedreactive group. In one embodiment, the PEG polymer is a linear orbranched polyether with a terminal hydroxyl group, such as a monomethoxyPEG, which is derivatized with a linking group, such as an amine,maleimide or carboxylic acid. The available reactive groups of theconstruct dictate the derivatized linking group employed on the PEGpolymer. Thus, in one embodiment, the N-terminal amine of the constructis employed, using a carboxylic acid derivatized PEG. In anotherembodiment, the C-terminal amine of the construct is employed, againusing a carboxylic acid derivatized PEG. In yet another embodiment, if aLys residue or homolog thereof is present in the construct, either the αor ε amino group thereof may be employed, again using a carboxylic acidderivatized PEG. Maleimide derivatized PEG may be employed with either areactive thiol or hydroxyl group on the construct. Similarly, aminederivatized PEG may be employed with a reactive carboxyl group on anyterminal group or side chain of an amino acid residue, on a Q group of asurrogate, on a J group of a surrogate, or on an R or R′ group of asurrogate.

Thus, in one aspect, PEG is activiated with one or more electrophilicgroups and may be employed for coupling to amino groups of theconstruct, including coupling to an E amino group of a side chain or anN-terminal or C-terminal amine. Representative electrophilic reactivegroups include succinimidyl α-methylbutanoate and other α-methylbutyricacid esters, as disclosed in U.S. Pat. Nos. 5,672,662 and 6,737,505, andmay be be used with proteins, as disclosed in U.S. Patent ApplicationPublication 2004/0235734. Alternatively, succinimidyl propionate may beemployed as a reactive group, as disclosed in U.S. Pat. No. 5,567,662,or N-hydroxysuccinimide may be employed with a branched PEG, asdisclosed in U.S. Pat. No. 5,932,462. The teachings of each of theforegoing patents and patent applications are incorporated by referenceas if set forth in full.

In another aspect, PEG polymers are provided with one or more reactivealdehyde groups, and employed for coupling to a terminal primary amine,such as an N-terminal or C-terminal amine. In another aspect, PEGpolymers are provided with one or more thiol-reactive groups, such as amaleimide, ortho-pyridyldisulfide, or thiol group, and are employed forcoupling to a reactive thiol in the construct of any of formula IIIthrough XIII, such as a reactive thiol in a cysteine side chain or areactive thiol in a Q group of a construct.

In one aspect, any of the methods, conjugates or schemes as disclosed inInternational Patent Publication No. WO 2004/047871, or any referencecited therein, may be employed with the constructs of this invention.The teaching of the foregoing patent applications is incorporated byreference as if set forth in full.

In general, some form of chemical modification may be employed to makean active PEG derivative with a reactive group. The reactive group maybe an active carbonate, an active ester, an aldehyde, or tresylate. Inpart, the reactive group of the PEG determines the amino acid terminalgroup or side chain moiety to which the PEG derivative is bound. Ingeneral, site specific PEGylation is preferred, in part because theresulting construct is homogeneous, minimizing loss of biologicalactivity and reducing immunogenicity.

In one embodiment, the PEG has a molecular weight of from about 200 MWto about 50,000 MW, more preferably from about 2,000 MW to about 20,000MW. In another embodiment, monomethoxy PEG, such as of the formulaCH₃—O(CH₂—CH₂—O)_(n)—CH₂—CH₂—OH or CH₃—O(CH₂—CH₂—O)_(n)—H, where n isany integer from 2 to about 1200, is employed, preferably derivatizedwith an amine, maleimide or carboxylic acid linking group.

In another embodiment, the prosthetic group, such as PEG, is conjugatedto a construct by means of an enzymatically labile linker as describedin Veronese F M and Pasut G. Pegylation, successful approach to drugdelivery. Drug Discovery Today 10:1451-1458 (2005), and the methodsdisclosed therein are incorporated here by reference.

In another embodiment, the prosthetic group employed is a polymer withboth a lipophilic moiety and a hydrophilic polymer moiety, as disclosedin U.S. Pat. Nos. 5,359,030 and 5,681,811. In a related embodiment, theprosthetic group employed is an oligomer conjugate with a hydrophiliccomponent, such as a PEG polymer, and a lipophilic component, such as abranched fatty acid or alkyl chain, linked by a hydrolyzable bond, suchas an ester bond, as disclosed in U.S. Pat. No. 6,309,633. In anotherrelated embodiment, the prosthetic group employed is an oligomer thatincludes poly(propylene glycol), and preferably at least twopoly(propylene glycol) subunits, as disclosed in U.S. Pat. No.6,858,580. The teachings of each of the foregoing patents and patentapplications are incorporated by reference as if set forth in full.

In yet another embodiment, the teachings of U.S. Published PatentApplication 2004/0203081 are incorporated here by reference, includingspecifically teachings relating to prosthetic groups, referred to insuch application as “modifying moieties,” attached to variousnatriuretic compounds, and specifically oligomeric structures having avariety of lengths and configurations. In a related embodiment, theteachings of International Patent Publication WO 2004/047871 areincorporated by reference, including teachings related to “modifyingmoieties” attached by means of “modifying moiety conjugation sites” tonatriuretic molecules binding to NPRA, it being understood that similarmethods could be employed with natriuretic molecules binding to othernatriuretic receptors.

Certain terms as used throughout the specification and claims aredefined as follows.

The “construct” and “amino acid residue sequences” of this invention canbe a) naturally-occurring, b) produced by chemical synthesis, c)produced by recombinant DNA technology, d) produced by biochemical orenzymatic fragmentation of larger molecules, e) produced by methodsresulting from a combination of methods a through d listed above, or f)produced by any other means for producing peptides or amino acidsequences.

By employing chemical synthesis, a preferred means of production, it ispossible to introduce various amino acids which do not naturally occurinto the construct, modify the N- or C-terminus, and the like, therebyproviding for improved stability and formulation, resistance to proteasedegradation, and the like, and to introduce one or more amino acidsurrogates into the construct.

The term “peptide” as used throughout the specification and claims isintended to include any structure comprised of two or more amino acids,including chemical modifications and derivatives of amino acids. Theamino acids forming all or a part of a peptide may be naturallyoccurring amino acids, stereoisomers and modifications of such aminoacids, non-protein amino acids, post-translationally modified aminoacids, enzymatically modified amino acids, and the like. The term“peptide” also includes dimers or multimers of peptides. A“manufactured” peptide includes a peptide produced by chemicalsynthesis, recombinant DNA technology, biochemical or enzymaticfragmentation of larger molecules, combinations of the foregoing or, ingeneral, made by any other method.

The term “amino acid side chain moiety” used in this invention,including as used in the specification and claims, includes any sidechain of any amino acid, as the term “amino acid” is defined herein.This thus includes the side chain moiety present in naturally occurringamino acids. It further includes side chain moieties in modifiednaturally occurring amino acids, such as glycosylated amino acids. Itfurther includes side chain moieties in stereoisomers and modificationsof naturally occurring protein amino acids, non-protein amino acids,post-translationally modified amino acids, enzymatically synthesizedamino acids, derivatized amino acids, constructs or structures designedto mimic amino acids, and the like. For example, the side chain moietyof any amino acid disclosed herein is included within the definition. A“derivative of an amino acid side chain moiety” as hereafter defined isincluded within the definition of an amino acid side chain moiety.

The “derivative of an amino acid side chain moiety” is a modification toor variation in any amino acid side chain moiety, including amodification to or variation in either a naturally occurring orunnatural amino acid side chain moiety, wherein the modification orvariation includes: (a) adding one or more saturated or unsaturatedcarbon atoms to an existing alkyl, aryl, or aralkyl chain; (b)substituting a carbon in the side chain with another atom, preferablyoxygen or nitrogen; (c) adding a terminal group to a carbon atom of theside chain, including methyl (—CH₃), methoxy (—OCH₃), nitro (—NO₂),hydroxyl (—OH), or cyano (—C≡N); (d) for side chain moieties including ahydroxy, thiol or amino groups, adding a suitable hydroxy, thiol oramino protecting group; or (e) for side chain moieties including a ringstructure, adding one or ring substituents, including hydroxyl, halogen,alkyl, or aryl groups attached directly or through an ether linkage. Foramino groups, suitable amino protecting groups include, but are notlimited to, Z, Fmoc, Boc, Pbf, Pmc and the like.

The “amino acids” used in embodiments of the present invention, and theterm as used in the specification and claims, include the knownnaturally occurring protein amino acids, which are referred to by boththeir common three letter abbreviation and single letter abbreviation.See generally Synthetic Peptides: A User's Guide, G. A. Grant, editor,W.H. Freeman & Co., New York (1992), the teachings of which areincorporated herein by reference, including the text and table set forthat pages 11 through 24. An “amino acid” includes conventional α-aminoacids and further includes β-amino acids, α, α-disubstituted amino acidsand N-substituted amino acids wherein at least one side chain is anamino acid side chain moiety as defined herein. An “amino acid” furtherincludes N-alkyl α-amino acids, wherein the N-terminus amino group has aC₁ to C₆ linear or branched alkyl substituent. It may thus be seen thatthe term “amino acid” includes stereoisomers and modifications ofnaturally occurring protein amino acids, non-protein amino acids,post-translationally modified amino acids, enzymatically synthesizedamino acids, derivatized amino acids, constructs or structures designedto mimic amino acids, and the like. Modified and unusual amino acids aredescribed generally in Synthetic Peptides: A User's Guide, cited above;Hruby V. J., Al-obeidi F., Kazmierski W., Biochem. J. 268:249-262(1990); and Toniolo C., Int. J. Peptide Protein Res. 35:287-300 (1990);the teachings of all of which are incorporated herein by reference. Inaddition, the following abbreviations, including amino acids andprotecting and modifying groups thereof, have the meanings given:

-   -   Abu—gamma-amino butyric acid    -   12-Ado—12-amino dodecanoic acid    -   Aib—alpha-aminoisobutyric acid    -   6-Ahx—6-amino hexanoic acid    -   Amc—4-(aminomethyl)-cyclohexane carboxylic acid    -   8-Aoc—8-amino octanoic acid    -   Bip—biphenylalanine    -   Boc—t-butoxycarbonyl    -   Bzl—benzyl    -   Bz—benzoyl    -   Cit—citrulline    -   Dab—diaminobutyric acid    -   Dap—diaminopropionic acid    -   Dip—3,3-diphenylalanine    -   Disc—1,3-dihydro-2-H-isoindolecarboxylic acid    -   Et—ethyl    -   Fmoc—fluorenylmethoxycarbonyl    -   Hept—heptanoyl(CH₃—(CH₂)₅—C(═O)—)    -   Hex—hexanoyl(CH₃—(CH₂)₄—C(═O)—)    -   HArg—homoarginine    -   HCys—homocysteine    -   HLys—homolysine    -   HPhe—homophenylalanine    -   HSer—homoserine    -   Me—methyl    -   Met(O)—methionine sulfoxide    -   Met(O₂)—methionine sulfone    -   Nva—norvaline    -   Pgl—phenylglycine    -   Pr—propyl    -   Pr-isopropyl    -   Sar—sarcosine    -   Tle—tert-butylalanine    -   z—benzyloxycarbonyl

In the listing of constructs according to the present invention,conventional amino acid residues have their conventional meaning asgiven in Chapter 2400 of the Manual of Patent Examining Procedure,8^(th) Ed. Thus, “Nle” is norleucine; “Asp” is aspartic acid; “His” ishistidine; “Arg” is arginine; “Trp” is tryptophan; “Lys” is lysine;“Gly” is glycine; “Pro” is proline; “Tyr” is tyrosine, “Ser” is serineand so on. All residues are in the L-isomer configuration unless theD-isomer is specified, as in “D-Ala” for D-alanine.

A single amino acid, including stereoisomers and modifications ofnaturally occurring protein amino acids, non-protein amino acids,post-translationally modified amino acids, enzymatically synthesizedamino acids, derivatized amino acids, an α, α-disubstituted amino acidderived from any of the foregoing (i.e., an α, α-disubstituted aminoacid wherein at least one side chain is the same as that of the residuefrom which it is derived), a β-amino acid derived from any of theforegoing (i.e., a β-amino acid which other than for the presence of aβ-carbon is otherwise the same as the residue from which it is derived)and the like, including all of the foregoing, is sometimes referred toherein as a “residue.”

An “α, α-disubstituted amino acid” includes any α-amino acid having afurther substituent in the α-position, which substituent may be the sameas or different from the side chain moiety of the α-amino acid. Suitablesubstituents, in addition to the side chain moiety of the α-amino acid,include C₁ to C₆ linear or branched alkyl. Aib is an example of an α,α-disubstituted amino acid. While α, α-disubstituted amino acids can bereferred to using conventional L- and D-isomeric references, it is to beunderstood that such references are for convenience, and that where thesubstituents at the α-position are different, such amino acid caninterchangeably be referred to as an α, α-disubstituted amino acidderived from the L- or D-isomer, as appropriate, of a residue with thedesignated amino acid side chain moiety. Thus(S)-2-Amino-2-methyl-hexanoic acid can be referred to as either an α,α-disubstituted amino acid derived from L-Nle or as an α,α-disubstituted amino acid derived from D-Ala. Whenever an α,α-disubstituted amino acid is provided, it is to be understood asincluding all (R) and (S) configurations thereof.

An “N-substituted amino acid” includes any amino acid wherein an aminoacid side chain moiety is covalently bonded to the backbone amino group,optionally where there are no substituents other than H in the α-carbonposition. Sarcosine is an example of an N-substituted amino acid. By wayof example, sarcosine can be referred to as an N-substituted amino acidderivative of Ala, in that the amino acid side chain moiety of sarcosineand Ala is the same, methyl.

The term “amino acid surrogate” includes a molecule disclosed hereinwhich is a mimic of a residue, including but not limited to piperazinecore molecules, keto-piperazine core molecules and diazepine coremolecules. Unless otherwise specified, an amino acid surrogate isunderstood to include both a carboxyl group and amino group, and a groupcorresponding to an amino acid side chain, or in the case of an aminoacid surrogate of glycine, no side chain other than hydrogen. Thus anamino acid surrogate includes a molecule of the general formula offormula I or II given above. An amino acid surrogate further includesmolecules of any of the following structures, it being understood thatfor convenience such structures are given as the isolated surrogate, notincluding any protecting group and not bound by one or two peptide bondsto one or two amino acid residues forming a part of a construct of theinvention:

where R, R′, x and the asterisks are as defined for the surrogate offormula I. An amino acid surrogate further includes molecules of any ofthe following structures, again it being understood that for conveniencesuch structures are given as the isolated surrogate, not including anyprotecting group and not bound by one or two peptide bonds to one or twoamino acid residues forming a part of a construct of the invention:

where R, R′, x and the asterisks are as defined for the surrogate offormula I. For purposes of synthesis, either the carboxyl group or theamino group of any amino acid surrogate is preferably protected by aprotecting group, such that it is not reactive while the protectinggroup is present, and similarly any reactive group forming a part of Ror R′ may similarly be protected by a protecting group. It will beappreciated that the surrogates of the present invention have more thanone asymmetric center, and therefore are capable of existing in morethan one stereoisomeric form. Some of the compounds may also exist asgeometric isomers and rotamers. Furthermore, some compounds of theinvention may also have conformational axial chirality resulting inatropisomers. The invention extends to each of these forms individuallyand to mixtures thereof, including racemates. In one aspect, surrogateisomers may be separated conventionally by chromatographic methods or byuse of a resolving agent. In another aspect, individual surrogateisomers, or enantiomerically pure surrogates, are prepared by syntheticschemes, such as those disclosed herein or variants of such schemes,employing asymmetric synthesis using chiral intermediates, reagents orcatalysts.

The term “C-terminus capping group” includes any terminal group attachedthrough the terminal ring carbon atom or, if provided, terminal carboxylgroup, of the C-terminus of a construct. The terminal ring carbon atomor, if provided, terminal carboxyl group, may form a part of a residue,or may form a part of an amino acid surrogate. In a preferred aspect,the C-terminus capping group forms a part of an amino acid surrogatewhich is at the C-terminus position of the construct. The C-terminuscapping group includes, but is not limited to, —(CH₂)_(n)—OH, —(CH₂),—C(═O)—OH, —(CH₂)_(m)—OH, —(CH₂), —C(═O)—N(v₁)(v₂),—(CH₂)_(n)—C(═O)—(CH₂)_(m)—N(v₁)(v₂), —(CH₂)_(n)—O—(CH₂)_(m)—CH₃,—(CH₂)_(n)—C(═O)—NH—(CH₂)_(m)—CH₃,—(CH₂)_(n)—C(═O)—NH—(CH₂)_(m)—N(v₁)(v₂),—(CH₂)_(n)—C(═O)—N—((CH₂)_(m)—N(v₁)(v₂))₂,—(CH₂)_(n)—C(═O)—NH—CH(—C(═O)—OH)—(CH₂)_(m)—N(v₁)(v₂),—C(═O)—NH—(CH₂)_(m)—NH—C(═O)—CH(N(v₁)(v₂))((CH₂)_(m)—N(v₁)(v₂)), or—(CH₂)_(n)—C(═O)—NH—CH(—C(═O)—NH₂)—(CH₂)_(m)—N(v₁)(v₂), including all(R) or (S) configurations of the foregoing, where v₁ and v₂ are eachindependently H, a C₁ to C₁₇ linear or branched alkyl chain, m is 0 to17 and n is 0 to 2; or any omega amino aliphatic, terminal aryl oraralkyl, including groups such as methyl, dimethyl, ethyl, propyl,isopropyl, butyl, isobutyl, pentyl, hexyl, allyl, cyclopropane methyl,hexanoyl, heptanoyl, acetyl, propionoyl, butanoyl, phenylacetyl,cyclohexylacetyl, naphthylacetyl, cinnamoyl, phenyl, benzyl, benzoyl,12-Ado, 7′-amino heptanoyl, 6-Ahx, Amc or 8-Aoc, or any single naturalor unnatural α-amino acid, β-amino acid or α, α-disubstituted aminoacid, including all (R) or (S) configurations of the foregoing,optionally in combination with any of the foregoing non-amino acidcapping groups. In the foregoing, it is to be understood that, forexample, —C(═O)—NH—(CH₂)_(m)—NH—C(═O)—CH(N(v₁)(v₂))((CH₂)_(m)—N(v₁)(v₂))is:

The term “N-terminus capping group” includes any terminal group attachedthrough the terminal amine of the N-terminus of a construct. Theterminal amine may form a part of a residue, or may form a part of anamino acid surrogate. In a preferred aspect, the N-terminus cappinggroup forms a part of an amino acid surrogate which is at the N-terminusposition of the construct. The N-terminus capping group includes, but isnot limited to, any omega amino aliphatic, acyl group or terminal arylor aralkyl including groups such as methyl, dimethyl, ethyl, propyl,isopropyl, butyl, isobutyl, pentyl, hexyl, allyl, cyclopropane methyl,hexanoyl, heptanoyl, acetyl, propionoyl, butanoyl, phenylacetyl,cyclohexylacetyl, naphthylacetyl, cinnamoyl, phenyl, benzyl, benzoyl,12-Ado, 7-amino heptanoyl, 6-Ahx, Amc or 8-Aoc, or alternatively anN-terminus capping group is —(CH₂)_(m)—NH(v₃), —(CH₂)_(m)—CH₃,—C(═O)—(CH₂)_(m)—CH₃, —C(═O)—(CH₂)_(m)—NH(v₃),—C(═O)—(CH₂)_(m)—C(═O)—OH, —C(═O)—(CH₂)_(m)—C(═O)-(v₄),—(CH₂)_(m)—C(═O)—OH, —(CH₂)_(m)—C(═O)-(v₄), C(═O)—(CH₂)_(m)—O(v₃),—(CH₂)_(m)—O(v₃), C(═O)—(CH₂)_(m)—S(v₃), or —(CH₂)_(m)—S(v₃), where v₃is H or a C₁ to C₁₇ linear or branched alkyl chain, and v₄ is a C₁ toC₁₇ linear or branched alkyl chain and m is 0 to 17.

A phenyl ring is “substituted” when the phenyl ring includes one or moresubstituents independently comprising hydroxyl, halogen, alkyl, or arylgroups attached directly or through an ether linkage. Where the phenylring is so substituted, the amino acid residue may be referred to assubstituted, as in substituted Phe, substituted HPhe or substituted Pgl.

The term “alkene” includes unsaturated hydrocarbons that contain one ormore double carbon-carbon bonds. Examples of alkene groups includeethylene, propene, and the like.

The term “alkenyl” includes a linear monovalent hydrocarbon radical oftwo to six carbon atoms or a branched monovalent hydrocarbon radical ofthree to six carbon atoms containing at least one double bond; examplesthereof include ethenyl, 2-propenyl, and the like.

The “alkyl” groups specified herein include those alkyl radicals of thedesignated length in either a straight or branched configuration.Examples of alkyl radicals include methyl, ethyl, propyl, isopropyl,butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl, isohexyl,and the like.

The term “alkynyl” includes a linear monovalent hydrocarbon radical oftwo to six carbon atoms or a branched monovalent hydrocarbon radical ofthree to six carbon atoms containing at least one triple bond; examplesthereof include ethynyl, propynal, butynyl, and the like.

The term “aryl” includes a monocyclic or bicyclic aromatic hydrocarbonradical of 6 to 12 ring atoms, and optionally substituted independentlywith one or more substituents selected from alkyl, haloalkyl,cycloalkyl, alkoxy, alkylthio, halo, nitro, acyl, cyano, amino,monosubstituted amino, disubstituted amino, hydroxy, carboxy, oralkoxy-carbonyl. Examples of aryl groups include phenyl, biphenyl,naphthyl, 1-naphthyl, and 2-naphthyl, derivatives thereof, and the like.

The term “aralkyl” includes a radical —R^(a)R^(b) where R^(a) is analkylene (a bivalent alkyl) group and R^(b) is an aryl group as definedabove. Examples of aralkyl groups include benzyl, phenylethyl,3-(3-chlorophenyl)-2-methylpentyl, and the like.

The term “aliphatic” includes compounds with hydrocarbon chains, such asfor example alkanes, alkenes, alkynes, and derivatives thereof.

The term “acyl” includes a group R—C(═O)—, where R is an organic group.An example is the acetyl group CH₃—C(═O)—, referred to herein as “Ac”.

A peptide or aliphatic moiety is “acylated” when an aryl, alkyl orsubstituted alkyl group as defined above is bonded through one or morecarbonyl {—(C═O)—} groups. A peptide is most usually acylated at theN-terminus.

An “omega amino aliphatic” includes an aliphatic moiety with a terminalamino group. Examples of omega amino aliphatics include7′-amino-heptanoyl and the amino acid side chain moieties of ornithineand lysine.

The term “heteroaryl” includes mono- and bicyclic aromatic ringscontaining from 1 to 4 heteroatoms selected from nitrogen, oxygen andsulfur. 5- or 6-membered heteroaryl are monocyclic heteroaromatic rings;examples thereof include thiazole, oxazole, thiophene, furan, pyrrole,imidazole, isoxazole, pyrazole, triazole, thiadiazole, tetrazole,oxadiazole, pyridine, pyridazine, pyrimidine, pyrazine, and the like.Bicyclic heteroaromatic rings include, but are not limited to,benzothiadiazole, indole, benzothiophene, benzofuran, benzimidazole,benzisoxazole, benzothiazole, quinoline, benzotriazole, benzoxazole,isoquinoline, purine, furopyridine and thienopyridine.

An “amide” includes compounds that have a trivalent nitrogen attached toa carbonyl group (—C(═O)—NH₂), such as for example methylamide,ethylamide, propylamide, and the like.

An “imide” includes compounds containing an imido group(—C(═O)—NH—C(═O)—).

An “amine” includes compounds that contain an amino group (—NH₂).

A “nitrile” includes compounds that are carboxylic acid derivatives andcontain a (—CN) group bound to an organic group.

The term “halogen” is intended to include the halogen atoms fluorine,chlorine, bromine and iodine, and groups including one or more halogenatoms, such as —CF₃ and the like.

The term “composition”, as in pharmaceutical composition, is intended toencompass a product comprising the active ingredient(s), and the inertingredient(s) that make up the carrier, as well as any product whichresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions of the present invention encompass anycomposition made by admixing a construct of the present invention and apharmaceutically acceptable carrier.

The term “EC₅₀” is intended to include the molar concentration of anagonist which produced 50% of the maximum possible response for thatagonist. By way of example, a construct which, at a concentration of 72nM, produces 50% of the maximum possible response for that construct asdetermined in a cGMP assay, has an EC₅₀ of 72 nM. Unless otherwisespecified, the molar concentration associated with an EC₅₀ determinationis in nanomoles (nM).

The term “Ki (nM)” is intended to include the equilibrium receptorbinding affinity representing the molar concentration of a competingcompound that binds to half the binding sites of a receptor atequilibrium in the absence of a competitor. In general, the Ki isinversely correlated to the affinity of the compound for the receptor,such that if the Ki is low, the affinity is high. Ki may be determinedusing the equation of Cheng and Prusoff (Cheng Y., Prusoff W. H.,Biochem. Pharmacol. 22: 3099-3108, 1973):

${Ki} = \frac{{EC}_{50}}{1 + \frac{\lbrack{ligand}\rbrack}{K_{d}}}$

where “ligand” is the concentration of ligand, which may be aradioligand, and K_(d) is an inverse measure of receptor affinity whichproduces 50% receptor occupancy. Unless otherwise specified, the molarconcentration associated with a Ki determination is nM.

The chemical naming protocol and structure diagrams used herein employand rely on the chemical naming features as utilized by the ChemDrawprogram (available from Cambridgesoft Corp., Cambridge, Mass.). Inparticular, certain compound names were derived from the structuresusing the Autonom program as utilized by Chemdraw Ultra or ISIS base(MDL Corp.). In general, structure diagrams do not depict hydrogen atomsassociated with carbon atoms other than in terminal groups and otherspecial circumstances.

Certain structure diagrams and drawings herein, such as those includedin Tables 1 and 2, depict constructs composed of amino acid surrogatesand amino acid residues, with the surrogates identified by structurediagrams and the amino acid residues identified by a three letterabbreviation. Unless otherwise specified, it is understood that the bondbetween the surrogate and residue, or between the residue and surrogate,or between a surrogate and residues on both the N-terminus andC-terminus side thereof, is a conventional peptide bond, —C(═O)—NH— or,in the case where the peptide bond is to the ring nitrogen on theN-terminus of the surrogate, —C(═O)—N—. In general, in the depiction ofsuch bonds the atoms of the amino acid surrogate are depicted (e.g.,—C(═O)— or —N), but atoms of the amino acid residue are not depicted.

Formulation and Utility

The constructs disclosed herein can be used for both medicalapplications and animal husbandry or veterinary applications. Typically,the construct, or a pharmaceutical composition including the construct,is used in humans, but may also be used in other mammals. The term“patient” is intended to denote a mammalian individual, and is so usedthroughout the specification and in the claims. The primary applicationsof this invention involve human patients, but this invention may beapplied to laboratory, farm, zoo, wildlife, pet, sport or other animals.

The constructs disclosed herein may be used for the treatment of anycondition, syndrome or disease for which induction of anti-hypertensive,cardiovascular, renal, and/or endocrine effects are desired. Thisincludes specifically any condition, syndrome or disease for which anative natriuretic peptide may be employed. Thus the constructsdisclosed herein may be employed to cause desired natriuresis, diuresisand/or vasodilation in a patient.

In one aspect, the constructs disclosed herein are used in the treatmentof early stage, such as class 1, congestive heart failure. In anotheraspect, the constructs disclosed herein are used in the treatment ofchronic or decompensated congestive heart failure. In another aspect,the constructs disclosed herein are used in the treatment of acutecongestive heart failure, including acutely decompensated congestiveheart failure of patients with dyspnea at rest or with minimal activity.

Salt Form of Constructs. The constructs of this invention may be in theform of any pharmaceutically acceptable salt. The term “pharmaceuticallyacceptable salts” refers to salts prepared from pharmaceuticallyacceptable non-toxic bases or acids including inorganic or organic basesand inorganic or organic acids. Salts derived from inorganic basesinclude salts of aluminum, ammonium, calcium, copper, ferric, ferrous,lithium, magnesium, manganic, manganous, potassium, sodium, zinc, andthe like. Particularly preferred are the ammonium, calcium, lithium,magnesium, potassium, and sodium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, and basic ionexchange resins, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,histidine, hydrabamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine,tromethamine, and the like.

When the construct of the present invention is basic, acid additionsalts may be prepared from pharmaceutically acceptable non-toxic acids,including inorganic and organic acids. Such acids include acetic,benzenesulfonic, benzoic, camphorsulfonic, carboxylic, citric,ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, malonic, mucic, nitric, pamoic, pantothenic,phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonicacid, trifluoroacetic acid, and the like. Acid addition salts of theconstructs of this invention are prepared in a suitable solvent from theconstruct and an excess of an acid, such as hydrochloric, hydrobromic,sulfuric, phosphoric, acetic, trifluoroacetic, citric, tartaric, maleic,succinic or methanesulfonic acid. The acetate salt form is especiallyuseful. Where the constructs of embodiments of this invention include anacidic moiety, suitable pharmaceutically acceptable salts may includealkali metal salts, such as sodium or potassium salts, or alkaline earthmetal salts, such as calcium or magnesium salts.

In addition, Applicants have advantageously discovered that certain saltforms of the peptide constructs of the invention, including pamoate,octanoate, decanoate, oleate, stearate, sodium tannate and palmitatesalt forms, have an increased circulation half-life, in some casesdoubled, versus the corresponding acetate salt form. These salt formsare particularly well-suited for administration by subcutaneousinjection or intramuscular injection, especially for chronic treatment,due to the reduced frequency of dosing that may be achieved as a resultof the longer half-lives. While not being limited by theory, it isbelieved the increased half-life is related to a decrease in solubilityin comparison to the acetate salt form. The increased half-life saltforms of the peptide constructs of the invention may be manufactured byany method including, for example, ion exchange, mixing a solution of anacetate salt form of a construct with disodium pamoate to form a pamoatesuspension, or use of the desired salt during the final purificationstep(s) in the manufacture of the constructs.

Pharmaceutical Compositions. Another embodiment of the present inventionprovides a pharmaceutical composition that includes a construct of thisinvention and a pharmaceutically acceptable carrier. The carrier may bea liquid formulation, and is preferably a buffered, isotonic, aqueoussolution. Pharmaceutically acceptable carriers also include excipients,such as diluents, carriers and the like, and additives, such asstabilizing agents, preservatives, solubilizing agents, buffers and thelike, as hereafter described.

The constructs of the several embodiments of the present invention maybe formulated or compounded into pharmaceutical compositions thatinclude at least one construct of this invention together with one ormore pharmaceutically acceptable carriers, including excipients, such asdiluents, carriers and the like, and additives, such as stabilizingagents, preservatives, solubilizing agents, buffers and the like, as maybe desired. Formulation excipients may include polyvinylpyrrolidone,gelatin, hydroxy cellulose, acacia, polyethylene glycol, manniton,sodium chloride and sodium citrate. For injection or other liquidadministration formulations, water containing at least one or morebuffering constituents is preferred, and stabilizing agents,preservatives and solubilizing agents may also be employed. For solidadministration formulations, any of a variety of thickening, filler,bulking and carrier additives may be employed, such as starches, sugars,fatty acids and the like. For topical administration formulations, anyof a variety of creams, ointments, gels, lotions and the like may beemployed. For most pharmaceutical formulations, non-active ingredientswill constitute the greater part, by weight or volume, of thepreparation. For pharmaceutical formulations, it is also contemplatedthat any of a variety of measured-release, slow-release or time-releaseformulations and additives may be employed, so that the dosage may beformulated so as to effect delivery of a construct of this inventionover a period of time. For example, gelatin, sodiumcarboxymethylcellulose and/or other cellulosic excipients may beincluded to provide time-release or slower-release formulations,especially for administration by subcutaneous and intramuscularinjection.

In general, the actual quantity of constructs administered to a patientwill vary between fairly wide ranges depending on the mode ofadministration, the formulation used, and the response desired.

In practical use, the constructs can be combined as the activeingredient in an admixture with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques. The carrier may takea wide variety of forms depending on the form of preparation desired foradministration, for example, oral, parenteral (including intravenous),urethral, vaginal, nasal, dermal, transdermal, pulmonary, deep lung,inhalation, buccal, sublingual, or the like. In preparing thecompositions for oral dosage form, any of the usual pharmaceutical mediamay be employed, such as, for example, water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like in thecase of oral liquid preparations, such as, for example, suspensions,elixirs and solutions; or carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents and the like in the case of oral solidpreparations such as, for example, powders, hard and soft capsules andtablets.

Because of their ease of administration, tablets and capsules representan advantageous oral dosage unit form. If desired, a compositionincluding a construct of this invention may be coated by standardaqueous or nonaqueous techniques. The amount of active construct in suchtherapeutically useful compositions is such that an effective dosagewill be obtained. In another advantageous dosage unit form, sublingualpharmaceutical compositions may be employed, such as sheets, wafers,tablets or the like. The active construct can also be administeredintranasally as, for example, by liquid drops or spray.

The tablets, pills, capsules, and the like may also contain a bindersuch as gum tragacanth, acacia, corn starch or gelatin; excipients suchas dicalcium phosphate; a disintegrating agent such as corn starch,potato starch or alginic acid; a lubricant such as magnesium stearate;and a sweetening agent such as sucrose, lactose or saccharin. When adosage unit form is a capsule, it may contain, in addition to materialsof the above type, a liquid carrier such as a fatty oil.

Various other materials may be utilized as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar or both. A syrup or elixir may contain, in additionto the active ingredient, sucrose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Constructs may also be administered parenterally. Solutions orsuspensions of these active peptides may be prepared in water suitablymixed with a surfactant such as hydroxy-propylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols and mixturesthereof in oils. These preparations may optionally contain apreservative to prevent the growth of microorganisms. Lyophilized singleunit formulations may also be utilized, which are reconstituted, such aswith saline, immediately prior to administration, and thus do notrequire a preservative.

The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powders,such as lyophilized formulations, for the extemporaneous preparation ofsterile injectable solutions or dispersions. In all cases, the form mustbe sterile and must be fluid to the extent that it may be administeredby syringe. The form must be stable under the conditions of manufactureand storage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, a polyol,for example glycerol, propylene glycol or liquid polyethylene glycol,suitable mixtures thereof, and vegetable oils.

Constructs as disclosed herein may be therapeutically applied by meansof nasal administration. By “nasal administration” is meant any form ofintranasal administration of any of the constructs of this invention.The constructs may be in an aqueous solution, such as a solutionincluding saline, citrate or other common excipients or preservatives.The constructs may also be in a dry or powder formulation.

In an alternative embodiment, constructs may be administered directlyinto the lung. Intrapulmonary administration may be performed by meansof a metered dose inhaler, a device allowing self-administration of ametered bolus of a construct of this invention when actuated by apatient during inspiration. Both dry powder inhalation and nebulizedaerosols may be employed.

According to another embodiment of the present invention, constructs ofthis invention may be formulated with any of a variety of agents thatincrease effective nasal absorption of drugs, including peptide drugs.These agents should increase nasal absorption without unacceptabledamage to the mucosal membrane. U.S. Pat. Nos. 5,693,608, 5,977,070 and5,908,825, among others, teach a number of pharmaceutical compositionsthat may be employed, including absorption enhancers, and the teachingsof each of the foregoing, and all references and patents cited therein,are incorporated by reference.

If in an aqueous solution, certain constructs of the present inventionmay be appropriately buffered by means of saline, acetate, phosphate,citrate, acetate or other buffering agents, which may be at anyphysiologically acceptable pH, generally from about pH 4 to about pH 7.A combination of buffering agents may also be employed, such asphosphate buffered saline, a saline and acetate buffer, and the like. Inthe case of saline, a 0.9% saline solution may be employed. In the caseof acetate, phosphate, citrate, acetate and the like, a 50 mM solutionmay be employed. In addition to buffering agents, a suitablepreservative may be employed, to prevent or limit bacteria and othermicrobial growth. One such preservative that may be employed is 0.05%benzalkonium chloride.

It is also possible and contemplated that the construct may be in adried and particulate form. In a preferred embodiment, the particles arebetween about 0.5 and 6.0 μm, such that the particles have sufficientmass to settle on the lung surface, and not be exhaled, but are smallenough that they are not deposited on surfaces of the air passages priorto reaching the lung. Any of a variety of different techniques may beused to make dry powder microparticles, including but not limited tomicro-milling, spray drying and a quick freeze aerosol followed bylyophilization. With micro-particles, the constructs may be deposited tothe deep lung, thereby providing quick and efficient absorption into thebloodstream. Further, with such approach penetration enhancers are notrequired, as is sometimes the case in transdermal, nasal or oral mucosaldelivery routes. Any of a variety of inhalers can be employed, includingpropellant-based aerosols, nebulizers, single dose dry powder inhalersand multidose dry powder inhalers. Common devices in current use includemetered dose inhalers, which are used to deliver medications for thetreatment of asthma, chronic obstructive pulmonary disease and the like.Preferred devices include dry powder inhalers, designed to form a cloudor aerosol of fine powder with a particle size that is always less thanabout 6.0 μm.

Microparticle size, including mean size distribution, may be controlledby means of the method of making. For micro-milling, the size of themilling head, speed of the rotor, time of processing and the likecontrol the microparticle size. For spray drying, the nozzle size, flowrate, dryer heat and the like control the microparticle size. For makingby means of quick freeze aerosol followed by lyophilization, the nozzlesize, flow rate, concentration of aerosoled solution and the likecontrol the microparticle size. These parameters and others may beemployed to control the microparticle size.

The constructs of this invention may be therapeutically administered bymeans of an injection, typically a deep intramuscular injection, such asin the gluteal or deltoid muscle, of a time release injectableformulation. In one embodiment, a construct of this invention isformulated with a PEG, such as poly(ethylene glycol) 3350, andoptionally one or more additional excipients and preservatives,including but not limited to excipients such as salts, polysorbate 80,sodium hydroxide or hydrochloric acid to adjust pH, and the like. Inanother embodiment, a construct of this invention is formulated with apoly(ortho ester), which may be an auto-catalyzed poly(ortho ester) withany of a variable percentage of lactic acid in the polymeric backbone,and optionally one or more additional excipients. In one embodiment poly(D,L-lactide-co-glycolide) polymer (PLGA polymer) is employed,preferably a PLGA polymer with a hydrophilic end group, such as PLGARG502H from Boehringer Ingelheim, Inc. (Ingelheim, Germany). Suchformulations may be made, for example, by combining a construct of thisinvention in a suitable solvent, such as methanol, with a solution ofPLGA in methylene chloride, and adding thereto a continuous phasesolution of polyvinyl alcohol under suitable mixing conditions in areactor. In general, any of a number of injectable and biodegradablepolymers, which are preferably also adhesive polymers, may be employedin a time release injectable formulation. The teachings of U.S. Pat.Nos. 4,938,763, 6,432,438, and 6,673,767, and the biodegradable polymersand methods of formulation disclosed therein, are incorporated here byreference. The formulation may be such that an injection is required ona weekly, monthly or other periodic basis, depending on theconcentration and amount of construct, the biodegradation rate of thepolymer, and other factors known to those of skill in the art.

Routes of Administration. If it is administered by injection, theinjection may be intravenous, subcutaneous, intramuscular,intraperitoneal or other means known in the art. The constructs of thisinvention may be formulated by any means known in the art, including butnot limited to formulation as tablets, capsules, caplets, suspensions,powders, lyophilized preparations, suppositories, ocular drops, skinpatches, oral soluble formulations, sprays, aerosols and the like, andmay be mixed and formulated with buffers, binders, excipients,stabilizers, anti-oxidants and other agents known in the art. Ingeneral, any route of administration by which the constructs of thisinvention are introduced across an epidermal layer of cells may beemployed. Administration means may thus include administration throughmucous membranes, buccal administration, oral administration, dermaladministration, inhalation administration, pulmonary administration,nasal administration, urethral administration, vaginal administration,and the like.

In one aspect, a construct of this invention is administered by means ofa time-release injectable formulation, such as a construct of thisinvention in a formulation with a PEG, poly(ortho ester) or PLGApolymer. In another aspect, a construct of this invention isadministered by means of an automated delivery device providingsubcutaneous delivery, either continuous or intermittent. Any of theforegoing methods and formulations are particularly applicable fortreatment of chronic conditions or syndromes, including chroniccongestive heart failure and particularly chronic decompensatedcongestive heart failure.

In one aspect, any construct of this invention may be administered bysubcutaneous administration, including all the methods disclosed in U.S.Pat. No. 6,586,396. In another aspect, a patient, particularly a patientwho is relatively compensated or is a patient with congestive heartfailure in an outpatient setting, may be administered a construct ofthis invention by methods and in doses as disclosed in U.S. PatentApplication Publication 2004/0077537 and International PatentApplication Publication WO 2003/079979. In another aspect, a patient maybe administered a construct of this invention by means of the methods asdisclosed in U.S. Patent Application Publication 2005/0113286. In yetanother aspect, a patient who has undergone myocardial injury may betreated for cardiac remodeling by means of the methods as disclosed inU.S. Patent Application Publication 2006/0019890.

A construct of this invention may also be administered by transdermaladministration, including by means of the delivery system, including theapparatus, and the methods as disclosed in U.S. Patent ApplicationPublication 2006/0034903. Similarly, the hydrogel formulations and solidstate formulations disclosed therein may be adapted for use with theconstructs of this invention.

Therapeutically Effective Amount. In general, the actual quantity of aconstruct of this invention administered to a patient will vary betweenfairly wide ranges depending upon the mode of administration, theformulation used, and the response desired. The dosage for treatment isadministration, by any of the foregoing means or any other means knownin the art, of an amount sufficient to bring about the desiredtherapeutic effect. Thus, a therapeutically effective amount includes anamount of a construct or pharmaceutical composition of this inventionthat is sufficient to induce a desired effect, including specifically ananti-hypertensive, cardiovascular, renal and/or endocrine effect. In oneaspect a therapeutically effective amount is an amount that results indesired natriuresis, diuresis and/or vasodilation.

In general, the constructs of this invention are highly active. Forexample, a construct can be administered at about 0.001, 0.01, 0.05,0.1, 0.5, 1, 5, 10 or 100 μg/kg body weight, depending on the specificconstruct selected, the desired therapeutic response, the route ofadministration, the formulation and other factors known to those ofskill in the art.

Combination Therapy

It is also possible and contemplated that constructs according toseveral embodiments of the present invention are used in combinationwith other drugs or agents, particularly in the treatment of congestiveheart failure.

According to another aspect of the present invention, a method fortreating congestive heart failure is provided. The method includesadministering to the patient having congestive heart failure atherapeutically effective amount of a construct as disclosed herein incombination with a therapeutically effective amount of another compoundthat is useful in the treatment of congestive heart failure, oralternative that is useful in extending the bioavailability of aconstruct of this invention in a patient. In one aspect, a patient maybe administered a construct of this invention in combination with adiuretic, such as by means of the methods and diuretics disclosed inU.S. Patent Application Publication 2004/0063630. Diuretics which may beemployed in combination include thiazide-, loop- and potassium-sparingdiuretics, including without limitation diuretics such ashydrochlorothiazide (Hydrodiuril®), chlorthalindone, furosemide(Lasix®), spironolactone (Aldactone®) and triamterine.

In another aspect of the present invention, a method for treatingcongestive heart failure is provided by administering a therapeuticallyeffective amount of a construct as disclosed herein in combination witha therapeutically effective amount of an anti-hypertensive agent otherthan a diuretic. Such anti-hypertensive agents include generally calciumchannel blockers (including dihydropyridines and non-dihydropyridines),sympatholytic agents, non-specific adrenergic blocking agents,α-adrenergic antagonists (including nonselective and selectiveα1-blocking agents), 1-blockers (including non-selective as well asselective blockers and those with intrinsic sympathomimetic activity),vasodilators (for treatment of resistant and emergent hypertension),angiotensin converting enzyme inhibitors and angiotensin II antagonists.Anti-hypertensive agents that may be employed in combination includemixed α and β antagonists such as labetolol (Normodyne®); vasodilatorssuch as hyralazine (Apresoline®), minoxidil (Loniten®), nitroprusside(Nipride®), or diazoxide (Hyperstat IV®); calcium blockers such asnifedipine (Adalat®), diltiazem (Cardizem®), or verapamil (Calan®);sympatholytics such as clonidine (Catapres®), methyldopa (Aldomet®),reserpine (Serpasil®), or guanethidine (Ismelin®); ACE inhibitors suchas captopril (Capoten®), enalapril (Vasotec®) or lisinopril (Prinivil®);α-adrenergic antagonists such as phentolamine (Regitine®) or prazosin(Minipress®); angiotensin II antagonists such as losartan (Cozaar®); orβ-adrenergic antagonists such as propranolol (Inderal®), nadolol(Corgard®), metoprolol (Lopressor®) or pindolol.

Other Indications

While a primary use of the constructs of the present invention is in thetreatment and amelioration of symptoms of congesive heart failure, acutekidney failure and renal hypertension, the constructs of the presentinvention may be employed in any treatment scheme or modality for whichnatriuretic, diuretic and/or vasodilator compounds provide a therapeuticbenefit. Thus, in one aspect, the constructs of the present inventionmay be employed as additives to peritoneal dialysis solutions, asdisclosed in U.S. Pat. No. 5,965,533. In another aspect, the constructsof the present invention may be employed in opthomologic applications,as disclosed in International Patent Publication WO 00/18422.

Synthetic Methods of Amino Acid Surrogates

The following examples of methods of synthesis of amino acid surrogatesof the invention are intended to be exemplary, and it is to beunderstood that variations thereon may be undertaken by one of skill inthe art, and such variations are intended to be included herein.

Synthesis of Ketopiperazine Scaffolds Mimicking Amino Acids withoutFunctionalized R Side Chain (Methods A and B)

The constructs were prepared by a variety of methods as described inMethods A and B.

Method A: The dipeptides (3) were formed by the mixed anhydride method,using Boc-serine (OBn)-OH (1), and an α-amino ester (2). The dipeptideswere obtained in high yields, and usually no purification was required.Reduction of both the methyl ester and the amide group was done usingdiborane-tetrahydrofuran, with the secondary amines protected to givethe di-Boc protected amino alcohol intermediates (4). Oxidation of thealcohols with pyridinium dichromate (PDC) with concomitant cyclizationgave the piperazine-2-ones (5) in one step. Benzyl ether removal byhydrogenation, followed by protecting group exchange gave the Fmocprotected piperazine-2-ones (6). Finally, the primary alcohol wasoxidized to the acid by any of a number of different methods (PDC, Jonesoxidation, ruthenium chloride-sodium periodate,2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO) oxidation) togive the final products (7).

Synthesis of2-(3-benzyloxy-2-tert-butoxycarbonylamino-propionylamino)-2-substitutedacetic acid methyl ester (3): To a solution of 10 mmol of Boc serinebenzyl ether (1) in 30 mL of dry tetrahydrofuran, kept at −20° C. undernitrogen, was added 22 mmol of triethylamine, followed by the slowaddition of 11.4 mmol of isobutylchloroformate. A white solidprecipitated out. The slurry was stirred for 15 minutes, and then 11.1mmol of α-amino ester (2) was added in one portion. The slurry wasstirred at −20° C. for 30 minutes, and then allowed to warm up to roomtemperature, stirred for 2 hours, and then concentrated to dryness. Themixture was then partitioned between 50 mL of ethyl acetate/30 mL of 1Nhydrochloric acid solution. The layers were separated, and the organiclayer washed with 1×20 mL of 1N hydrochloric acid, and 1×20 mL ofsaturated sodium bicarbonate solution, dried over magnesium sulfate andconcentrated. Compounds (3) were usually obtained in yields above 90%,and no purification was required.

R Analytical Data for Compounds (3)

¹H NMR δ (CDCl₃): 1.43 (s, 9H, ^(t)Bu), 3.0-3.18 (two sets of dd, 2H,CH₂-Ph), 3.50-3.57 (t, 1H, CH₂O), 3.68 (s, 3H, CH₃O), 3.87-3.96 (br. d,1H, CH₂O), 4.23-4.33 (br. m, 1H, CHN), 4.45-4.57 (dd, 2H, CH₂O),4.80-4.88 (m, 1H, CHN), 5.30-5.37 (m, 1H, NH), 7.0-7.38 (a series of m,10H, Ph), yield = 96%, t_(R) = 6.88 min, (M⁺ + 1) = 456.99

¹H NMR δ (CDCl₃): 0.81-0.96 (a series of m, 6H, CH₃), 1.00-1.16 (m, 1H,CH₂), 1.30-1.45 (m, 1H, CH₂), 1.45 (s, 9H, ^(t)Bu), 1.80-1.96 (m, 1H,CH), 3.54-3.64 (dd, 1H, CH₂O), 3.70 (s, 3H, CH₃O), 3.82-3.96 (dd, 1H,CH₂O), 4.28-4.40 (m, 1H, CHN), 4.51-4.61 (m, and s, 3H, CH₂O, and CHN),5.51-5.61 (br. d, 1H, NH), 7.12-7.37 (br. m, 5H, Ph), yield = quant.,t_(R) = 6.93 min, (M⁺ + 1) = 423.25

¹H NMR δ (CDCl₃): 1.45 (s, 9H, ^(t)Bu), 3.73 (s, 3H, CH₃O), 3.84-3.90(m, 2H, CH₂N), 4.01-4.17 (m, 2H, CH₂O), 4.32-4.38 (br. m, 1H, CHN),4.54-4.58 (d, 2H, CH₂O), 5.46-5.57 (d, 1H, NH), 7.05-7.12 (br. m, 1H,Ph), 7.24-7.40 (m, 4H, Ph), yield = quant., t_(R) = 5.51 min, (M⁺ + 1) =367.07

Synthesis ofDi-Boc-2-substituted-(2-amino-3-benzyloxy-propyl-amino)-ethanol (4): Toa solution of 35 mmol of (3) in 50 mL of dry tetrahydrofuran, kept atroom temperature under nitrogen, was added 200 mL of 1N diboranesolution in tetrahydrofuran. The solution was stirred at roomtemperature overnight, and then slowly poured over an ice-cold solutionof 200 mL of 1N hydrochloric acid solution. The biphasic solution wasthen neutralized with solid sodium hydroxide. 140 mL of a saturatedsolution of sodium bicarbonate was added, followed by 70 mmol ofdi-tert-butyl-dicarbonate, and the mixture stirred for 2 days at roomtemperature, diluted with 200 mL of ethyl acetate and the layersseparated. The organic layer was dried over magnesium sulfate, andconcentrated. The products (4) were purified by silica gel columnchromatography.

R Analytical Data for Compounds (4)

¹H NMR δ (CDCl₃): 1.42 (s, 9H, ^(t)Bu), 1.48 (s, 9H, ^(t)Bu), 2.48-3.02(a series of m, 2H, CH₂-Ph), 3.1-3.48 (br. m, 1H, CH₂O), 3.25-3.48 (br.m, 2H, CH₂N), 3.50-3.75 (m, 2H, CH₂O), 3.80-3.97 (m, 2H, CH₂O, and CHN),4.25 (br. m, 1H, CHN), 4.45 (s 2H, CH₂O), 4.9 (br. s, 1H, OH), 5.3 (br.s, 1H, NH), 7.1-7.4 (m, 10H, Ph), yield = 76%, t_(R) = 8.04 min,(M⁺ + 1) = 515.25

¹H NMR δ (CDCl₃): 0.84-0.96 (m, CH, CH₂, CH₃), 1.42 (s, 9H, ^(t)Bu),1.45 (s, 9H, ^(t)Bu), 1.42-1.44 (m, 1H, CH), 2.88-3.11 (br. m, 2H,CH₂N), 3.42-3.57 (m, 2H, CH₂O), 3.62-4.10 (two m, 4H, CH₂O, and CHN),4.51 (s, 2H, CH₂O), 7.27-7.38 (m, 5H, Ph), yield = 80%, t_(R) = 8.19min, (M⁺+ 1) = 481.26

¹H NMR δ (CDCl₃): 1.35-1.43 (m, 18H, ^(t)Bu), 3.20-3.32 (m, 1H, CH₂N),3.55-3.84 (a series of m, 8H, CH₂N, CH₂O), 3.90-4.05 (m, 1H, CHN), 4.45(s, 2H, CH₂O), 4.9-5.02 (m, 1H, NH), 7.2-7.35 (m, 5H, Ph), yield = 56%,t_(R) = 6.40 min, (M⁺ + 1) = 425.21

Synthesis of 1,4-di-Boc-6-benzyloxymethyl-3-substituted-piperazin-2-one(5): A solution of 70 mmol of (4), and 400 mmol of pyridinium dichromatein 300 mL of dry dimethylformamide was stirred at 48° C. under nitrogenfor 6 hours, cooled to room temperature, poured into 1500 mL of water,and extracted with 4×500 mL of ethyl ether. The ethereal layers werecombined, dried over magnesium sulfate, and concentrated. The products(5) were purified by silica gel column chromatography.

R Analytical Data for Compounds (5)

¹H NMR δ (CDCl₃): 1.4 (s, 9H, ^(t)Bu), 1.5 (s, 9H, ^(t)Bu), 3.05- 3.58(a series of m, CH₂-Ph, and CH₂N), 4.1-4.32 (a series of m, 2H, CH₂N),4.47 (s, 2H, CH₂O), 4.78-4.86 (br. m, 1H, CHN), 7.12-7.42 (m, 10H, Ph),yield = 42%, t_(R) = 8.65 min, (M⁺ + 1) = 511.05.

¹H NMR δ (CDCl₃): 0.82-1.56 (four s, and four m, 27H, ^(t)Bu, CH, CH₂,and CH₃), 3.20-3.52 (a series of m, 2H, CH₂N), 3.60-3.88 (a series of m,2H, CH₂O), 4.20-4.60 (a series of m, one s, 4H, CH₂O, CHN), 7.21-7.37(m, 5H, Ph), yield = 24%, t_(R) = 9.23 min, (M⁺ + 1) = 477.32.

Synthesis of 4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-one (6): Asuspension of 19 mmol of (5) and 2 g of 10% palladium on carbon in 200mL of ethanol was hydrogenated at room temperature and atmosphericpressure overnight. The suspension was filtered through celite, andconcentrated. The residue was redissolved in 40 mL of 50%trifluoroacetic acid in dichloromethane. The solution was stirred atroom temperature for 2 hours, and then concentrated. The residue wasredissolved in 60 mL of tetrahydrofuran/40 mL of water, and neutralizedwith solid sodium bicarbonate, followed by the addition of 40 mmol ofsolid sodium bicarbonate, and 20 mmol of Fmoc chloride. The mixture wasthen stirred at room temperature for 2 hours, diluted with 300 mL ofethyl acetate, and the layers separated. The organic layer was driedover magnesium sulfate, concentrated, and purified by silica gel columnchromatography.

R Analytical Data for Compound (6)

¹H NMR δ (CDCl₃): 2.15-2.32 (br. m, 1H, CH₂—Ph), 2.70- 2.81 (br. m, 1H,CH₂-Ph), 3.0-3.32 (br. m, 3H, CHN, and CH₂N), 3.47-3.65 (br. m, 3H,CH₂O, and CHN), 3.95-4.22 (two m, 2H, CH, and CHN), 4.32-4.48 (br. m,2H, CH₂O), 4.84-4.92 (br. m, 1H, NH), 6.73-6.83 (br. m, 1H, Ph), 6.92-7.01 (br. m, 1H, Ph), 7.08-7.82 (a series of m, 11H, Ph, and fulvene),yield = 65%, t_(R) = 5.78 min, (M⁺ + 1) = 443.07.

¹H NMR δ (CDCl₃): 0.6-1.15 (br. peaks, 7H, CH₂, and CH₃), 1.20-1.42 (br.m, 1H, CH₂), 1.72-2.02 (two br. peaks, 1H, CH), 2.74-2.86 (t, 1/2H,CHN), 2.74-3.74 (a series of br. peaks, 5H, CH₂O, CH₂N, and CHN),4.16-4.22 (br. m, 1H, CH), 4.52-4.74 (br. m, 2H, CH₂O), 7.24-7.82 (aseries of m, 8H, fulvene), yield = 34%, t_(R) = 5.72 min, (M⁺ + 1) =408.95

¹H NMR δ (CDCl₃): 0.73-1.00 (m, 7H, CH₃), 2.2-2.3 (br. m, 0.5H, CH),2.74-4.62 (a series of br. peaks, 12H, CH₂N, CH₂O and CHN), 3.68 (s, 3H,CH₃O), 7.26-7.77 (m, 9H, fulvene), yield = 45% (3 steps), t_(R) = 5.34min, (M⁺ + 1) = 394.93

Synthesis of 4-Fmoc-5-substituted-6-oxo-piperazine-2-carboxylic acid(7): Compounds (7) were prepared by several methods.

(a) To a biphasic solution of 10 mmol of (6) in 180 mL of acetonitrile,180 mL of carbon tetrachloride, and 240 mL of water, cooled to 0° C.,was added 112 mmol of solid sodium periodate, followed by 340 mg ofruthenium chloride. The reaction was allowed to warm up to roomtemperature, stirred for 2 hours, and then filtered through celite. Thelayers were separated, and the aqueous layer re-extracted with 2×75 mLof ethyl acetate. The organic layers were combined, dried over magnesiumsulfate, and concentrated.

(b) A solution of 12 mmol of (6), and 59 mmol of PDC in 60 mL of drydimethylformamide was stirred at 48° C. under nitrogen for ˜5 hours,cooled to room temperature, and poured over 600 mL of water, andextracted with 3×200 mL of dichloromethane. The organic layers werecombined, dried over magnesium sulfate, and concentrated.

(c) To a solution of 17 mmol of (6) in 350 mL of acetone kept at roomtemperature was added 25 mL of Jones reagent (prepared from 8.0 g ofchromic acid, 17.4 mL of water, and 6.9 mL of concentrated sulfuricacid). The mixture was stirred for 1 hour, 150 mL of isopropanol wasadded, and the mixture filtered through celite. The celite was washedwith ethyl acetate. The organic layers were combined and concentrated.The residue was dissolved in 250 mL of ethyl acetate and washed with2×50 mL of water, dried over magnesium sulfate, and concentrated.

(d) To a solution of 81 mmol alcohol (6) in 810 mL of acetonitrile keptat room temperature, was added phosphate buffer solution (prepared with7.2 g of sodium phosphate monobasic, and 14.3 g of sodium phosphatedibasic in 295 mL of water), followed by the addition of 3.3 g (20.7mmol) of TEMPO, and 18.6 g (164.4 mmol) of sodium chlorite, and thebiphasic solution placed in an oil bath kept at 43° C., and then asolution of 43.3 mL (25.9 mmol) of sodium hypochlorite solution(prepared by mixing 19.3 mL of 10-13% sodium hypochlorite solution, and24 mL of water) was added slowly. The reaction was stirred at 43° C. for4 hours. The solution was cooled to room temperature, and a solution of200 mL of 10% sodium hydrogen sulfite solution was added, stirred for 10minutes, diluted with 500 mL of ethyl acetate, and the layers separated.The organic layer was washed with 1×100 mL of brine, 1×160 mL of 1Nhydrochloric acid solution, dried over sodium sulfate, and concentrated.

The products (7) were purified by silica gel column chromatography.

R Analytical Data for Compounds (7)

¹H NMR δ (CDCl₃): 2.36-2.45 (dd, 1H, CH₂-Ph), 2.62-2.76 (m, 1/2H,CH₂-Ph), 2.82-2.98 (m, 1/2H, CH₂-Ph), 3.13-3.25 (m, 1H, CH₂N), 3.98-4.64(a series of m, 6H, CHN, CH₂O, CH₂, and CH), 4.87 (br. m, 1/2H, NH),6.85 (br. s, 1H, Ph), 7.0-7.40 (a series of m, 12H, Ph and fulvene),9.18-9.40 (br. d, 1H, CO₂H), t_(R) = 5.91 min, (M⁺ + 1) = 457.37.

¹H NMR δ (CDCl₃): 0.64-1.02 (overlapping t, 6H, CH₃), 1.02-1.68 (threebr. m, 2H, CH₂), 1.96-2.16 (br. m, 1H, CH), 2.88-3.18 (m, 1H, CH₂N),3.85-4.12 (three m, 2H, CH₂N, and CHN), 4.18-4.35 (m, 1H, CH), 4.55-4.72(m, 2H, CH₂), 4.75-4.86 (br. m, 1H, NH), 7.28-7.82 (a series of m, 8H,fulvene), 9.1-9.26 (two br. s, 1H, CO₂H), t_(R) = 5.86 min, (M⁺ + 1) =423.20.

¹H NMR δ (CDCl₃): 0.62-1.03 (m, 7H, CH₃), 1.90-2.05 (br. m, 1H, CH),2.87-4.60 (a series of br. peaks, 8H, CH₂N, CH₂O and CHN and CH),7.29-7.80 (m, 9H, fulvene), yield = 61%, t_(R) = 5.52 min, (M⁺ + 1) =409.11

Method B: IntermediatesDi-Boc-2-substituted-(2-amino-3-benzyloxy-propyl-amino)-ethanols (4),prepared as described in method A, were oxidized to the acid usingTEMPO/isocyanuric acid reagent, and then esterified with iodomethane togive fully protected reduced dipeptide analogs (8). Deprotection of theBoc groups, and the benzyl ether, gave 3-substituted5-hydroxymethyl-piperazin-2-ones, which were protected as the Fmocderivatives to give (6), which were oxidized to the final product asdescribed in method A.

Synthesis of Di-Boc-(2-amino-3-benzyloxy-propylamino)-2-substitutedacetic acid methyl ester (8): To a suspension of 76 mmol of (4) in 680mL of acetone, and 210 mL of a saturated sodium bicarbonate solution,kept at 0° C., was added 21 mmol of solid sodium bromide, and 2.9 mmolof TEMPO, followed by the slow addition of 156 mmol oftrichloroisocyanuric acid. The reaction was stirred for 30 minutes at 0°C., and then at room temperature overnight, acidified with a solution of1N hydrochloric acid, and extracted with 2×300 mL of ethyl acetate. Theorganic layer was washed with 3×50 mL of 1N hydrochloric acid, driedover magnesium sulfate, and concentrated. The residue was redissolved in40 mL of dry dimethylformamide and 95 mmol of solid sodium bicarbonate,and 112 mmol of iodomethane was added, and the mixture stirred at roomtemperature under nitrogen until HPLC showed the reaction was complete;the solution was then diluted with 200 mL of ethyl ether, and washedwith 2×100 mL of water, dried over magnesium sulfate, and concentrated.The products (8) were purified by silica gel column chromatography.

R Analytical Data for Compounds (8)

¹H NMR δ (CDCl₃): 1.41 (s, 9H, ^(t)Bu), 1.46 (s, 9H, ^(t)Bu), 2.44-2.58(d, 1/2H, CH₂-Ph), 2.66-2.88 (d, 1/2H, CH₂-Ph), 3.16-3.46 (three sets ofm, 5H, CH₂-Ph, CH₂N, and CH₂O), 3.72 (s, 3H, CH₃O), 3.75-4.05 (two m,1H, CHN), 4.42 (s, 2H, CH₂O), 4.95-5.10 (d, 1/2H, NH), 5.30-5.38 (d,1/2H, NH), 7.10-7.38 (m, 10H, Ph), yield = 62%, t_(R) = 7.75 min,(M⁺ + 1) = 529.03.

¹H NMR δ (CDCl₃): 1.41 (s, 9H, ^(t)Bu), 1.42 (s, 9H, ^(t)Bu), 3.30-3.60(br. m, 4H, CH₂N, CH₂O), 3.70 (s, 3H, CH₃O), 3.75-3.95 (m, 2H, CH₂N),4.51 (s, 2H, CH₂O), 5.0-5.08 (br. s, 1H, NH), 7.25-7.37 (m, 5H, Ph),yield = 47% t_(R) = 7.28 min, (M⁺ + 1) = 453.17.

Synthesis of 4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-one (6): Asolution of 36 mmol of (8) in 40 mL of 50% trifluoroacetic acid indichloromethane was stirred at room temperature for 2 hours, and thenpoured in 200 mL of saturated sodium bicarbonate solution. The layerswere separated, and the organic layer concentrated. The residue wasredissolved in 100 mL of ethyl acetate, and washed with 2×50 mL ofwater, dried over magnesium sulfate, and concentrated. The residue wasdissolved in 100 mL of ethanol, and 5 g of 10% palladium on carbon and35 mL of a 1N hydrochloric acid solution was added, and the mixturehydrogenated at room temperature and atmospheric pressure until HPLCshowed the reaction was complete; the solution was then filtered throughcelite and concentrated. The residue was redissolved in 80 mL of ethylacetate, 70 mmol of sodium bicarbonate in 30 mL of water was added, andthe mixture stirred at room temperature overnight. The ethyl acetate wasremoved and 100 mL of tetrahydrofuran was added, followed by 178 mmol ofsolid sodium bicarbonate and 43 mmol of Fmoc chloride, and the mixturewas stirred until HPLC showed it was complete, diluted with 300 mL ofethyl acetate, and the layers separated. The organic layer was washedwith 2×50 mL of water, dried over magnesium sulfate, and concentrated.The products (6) were purified by silica gel column chromatography.

Synthesis of 4-Fmoc-5-substituted-6-oxo-piperazine-2-carboxylic acid(7): Compounds (7) were prepared as described in method A.

General Common Synthetic Scheme for the Preparation of KetopiperazineScaffolds Applicable to Compounds With or Without Functionalized Rsidechains (Methods C, E, F)

Method C: (2-Fmoc-amino-3-R′—O-propylamino)-2-substituted acetic acidmethyl esters (10) were prepared by reductive amination of FmocO-protected serinal (9) with α-amino esters (2), using either sodiumcyanoborohydride or sodium triacetoxyborohydride as the reducing agent.The Fmoc O-protected serinal (9) required for the reductive aminationwas prepared according to method D, either by reduction of the ester(12) by di-isobutylaluminun hydride, by oxidation of Fmoc O-protectedserinol (13) with Dess-Martin periodinane, or by reduction of the FmocO-protected serine Weinreb amide (14) with lithium aluminum hydride. Thepreferred method for the preparation of Fmoc O-protected serinals (9)was the reduction of the Weinreb amide analog.(2-Fmoc-amino-3-R′—O-propylamino)-2-substituted acetic acid methylesters (10) were then N and O deprotected, cyclized, and Fmoc protectedto give 3-substituted 6-hydroxymethyl-piperazin-2-ones (6), which werethen oxidized to the final product as described in method A.

The protecting group (R′) on the hydroxyl group of Fmoc-O-protectedserinal (9) used in the synthesis depends on the nature of the sidechain R of the amino ester. When R contained no functional groups, theside chain of Fmoc serine was protected as the ^(t)Bu ether. When Rcontained functional groups, the side chain of Fmoc serine was protectedas the trityl ether.

Method D: Synthesis of various Fmoc-O-protected serinals (9). Synthesisof Fmoc-O—R′ serine methyl ester (12): A slight suspension of 80 mmol ofFmoc O—R′ serine (11), 10.0 g (120 mmol) of solid sodium bicarbonate,and 10.0 mL (160 mmol) of iodomethane in 80 mL of dry dimethylformamide,kept under nitrogen, was stirred at room temperature overnight. Thereaction mixture was then poured over 500 mL of water, and the solidfiltered. The solid was redissolved in 800 mL of ethyl acetate, andwashed with 1×200 mL of water, dried over magnesium sulfate, andconcentrated. No purification was required.

R′ Analytical Data for Compounds (12) ^(t)Bu ¹H NMR δ (CDCl₃): 1.14 (s,9H, ^(t)Bu), 3.57-3.70 (m, 1H, CH₂—O), 3.75 (s, 3H, O—CH₃), 3.79-3.83(m, 1H, CH₂—O), 4.01-4.50 (a series of multiples, 4H), 5.64-5.68 (d, 1H,NH), 7.28-7.78 (8H, fulvene), yield = 93% t_(R) = 7.8 min. Trt ¹H NMR δ(CDCl₃): 3.42-3.48 (m, 1H, CH₂—O), 3.59-3.66 (m, 1H, CH₂—O), 3.81 (s,3H, CH₃—O), 4.10-4.18 (m, 1H, CH), 4.36-4.42 (m, 2H, CH₂—O), 4.50-4.57(m, 1H, CH—N), 5.73-5.78 (d, 1H, NH), 7.22-7.82 (8H, fulvene), yield =quant., t_(R) = 9.04 min.

Synthesis of Fmoc-O—R′ serinol (13): To a solution of 10.0 mmol of FmocO—R′ serine (11) in 50 mL of dry tetrahydrofuran, kept at −20° C. undernitrogen, was added 1.77 mL (12.7 mmol) of triethyl amine, followed bythe slow addition of 1.57 mL (12.0 mmol) of isobutylchloroformate. Themixture was stirred for 30 minutes, and then poured slowly over anice-cold solution of 3.77 g (99.6 mmol) of sodium borohydride in 10 mLof water, keeping the temperature below 5° C. The reaction was stirredat 0° C. for 15 minutes, and then quenched with 1N hydrochloric acidsolution. The reaction mixture was diluted with 100 mL of ethyl acetate,and the layers separated. The organic layer was washed with 2×25 mL of1N hydrochloric acid solution, 2×25 mL of water, dried over magnesiumsulfate and concentrated. The compounds were purified by silica gelcolumn chromatography.

R′ Analytical Data for Compounds (13) ^(t)Bu ¹H NMR δ (CDCl₃): 1.14 (s,9H, ^(t)Bu), 2.90-2.95 (d, 1/2H, CH₂—O), 3.63 (d, 2H, CH₂—O), 3.65-3.93(m, 3H, CH₂—O), 4.20-4.35 (t, 1H, CH), 4.35-4.45 (d, 2H, CH₂), 5.50-5.57 (d, 1H, NH), 7.26-7.8 (8H, fulvene), yield = 85%, t_(R) = 6.42 min.Trt ¹H NMR δ (CDCl₃): 3.24-3.32 (br. d, 1H, CH₂—O), 3.30-3.45 (br. m,1H, CH₂—O), 3.60- 3.987 (br. m, 3H, CH₂—O, and CH—N), 4.13-4.22 (br. m,1H, CH), 4.32-4.40 (br. d, 2H, CH₂), 5.24-5.32 (br. d, 1H, NH),7.16-7.76 (23H, fulvene, and Trt), yield = 92%, t_(R) = 8.39 min.

Synthesis of Fmoc-O—R′ serine Weinreb amide (14): A suspension of 20.2mmol of Fmoc O—R′ serine (11), 6.98 g (21.6 mmol) of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU), and 2.5 mL (22.7 mmol) of N-methyl-morpholine in 80 mL of drydichloromethane was stirred at room temperature under nitrogen for 20minutes, and then 3.02 g (31 mmol) of N,O-di-methyl-hydroxylaminehydrochloride and 3.3 mL (30 mmol) of N-methyl-morpholine were added,and the suspension stirred at room temperature overnight. The solutionformed was then concentrated to dryness, repartitioned between 200 mL ofethyl acetate and 100 mL of water, washed with 2×40 mL of 1Nhydrochloric acid solution and then 2×40 mL of saturated sodiumbicarbonate solution, dried over magnesium sulfate, and concentrated. Nopurification was required.

R′ Analytical Data for Compounds (14) ^(t)Bu ¹H NMR δ (CDCl₃): 1.45 (s,9H, ^(t)Bu), 3.30 (s, 3H, CH₃—N), 3.55-3.7 (m, 2H, CH₂—O), 3.76 (s, 3H,CH₃—O), 4.19-4.26 (m, 1H, CH), 4.30-4.38 (m, 2H, CH₂—O), 4.82-4.91(broad m, 1H, CHN), 5.68-5.75 (d, 1H, NH), 7.2-7.8 (8H, fulvene), yield= quant., t_(R) = 6.59 min. Trt ¹H NMR δ (CDCl₃): 3.24 (s, 3H, CH₃N),3.34-3.46 (m 2H, CH₂O), 3.62 (s, 3H, CH₃O), 4.15-4.37 (two m, CH₂, CH),4.86-4.98 (m 1H, CHN), 5.80-5.86 (d, 1H, NH), 7.18-7.8 (a series of m,23H, Trt and fulvene), yield = quant., t_(R) = 8.0 min.

Synthesis of Fmoc-O—R′ serinal (9) from ester (12): To a solution of 3.5mmol of (12) in 5 mL of tetrahydrofuran, kept at −78° C. under nitrogen,was added slowly 10 mL of 1N diisobutyl aluminum hydride (DIBAL)solution, stirred for 15 minutes, and quenched by the slow addition of asaturated solution of sodium and potassium tartrate. The reaction wasallowed to warm up to room temperature, diluted with 50 mL of ethylacetate, and 50 mL of a saturated solution of sodium and potassiumtartrate was added. The layers were separated, and the aqueous layerre-extracted with 1×50 mL of ethyl acetate. The organic layers werecombined, dried over magnesium sulfate, and concentrated. Compounds (9)were used without further purification in the next step.

R′ Analytical Data for Compounds (9) ^(t)Bu ¹H NMR δ (CDCl₃): 1.16 (s,9H, ^(t)Bu), 3.59-3.66 (dd, 1H, CH₂O), 3.90-3.98 (dd, 1H, CH₂O),4.20-4.27 (t, 1H, CH), 4.32-4.45 (two m, 3H, CHN, and CH₂O), 5.64-5.74(br. d, 1H, NH), 7.28-7.35 (m, 2H, fulvene), 7.36-7.44 (m, 2H, fulvene),7.58-7.65 (d, 2H, fulvene), 7.73-7.78 (d, 2H, fulvene), 9.62 (s, 1H,CHO). Trt ¹H NMR δ (CDCl₃): 3.53-3.61 (dd, 1H, CH₂O), 3.66-3.75 (dd, 1H,CH₂O), 4.33-4.47 (two m, 4H, CHN, CH, and CH₂), 5.66-5.75 (d, 1H, NH),7.20-7.81 (a series of m, 23H, Trt, and fulvene), 9.6 (s, 1H, CHO).

Synthesis of Fmoc-O—R′ serinal (9) from alcohol (13): To a solution of80 mmol of Fmoc-O—R′ serinol (13) in 200 mL of dry dichloromethane, keptat room temperature under nitrogen, was added 88 mmol of Dess-Martinperiodinane, and the reaction was stirred for 2.5 hours and quenched byaddition of 400 mL of 10% aqueous sodium thiosulfate solution. Thelayers were separated, and the organic layer concentrated, diluted with300 mL of ethyl ether, and washed three times with a saturated aqueousbicarbonate solution containing 10% sodium thiosulfate, dried overmagnesium sulfate, and concentrated.

Synthesis of Fmoc-O—R′ serinal (9) from Weinreb amide (14): To asolution of 8.8 g (20.2 mmol) of crude Fmoc-O—R′ serine Weinreb amideintermediate (14) in 60 mL of dry tetrahydrofuran, cooled to −78° C.under nitrogen, was added 30 mL of 1N lithium aluminum hydride solutionin tetrahydrofuran. The solution was stirred for 15 minutes and thenquenched by the slow addition of 30 mL of a 1.4N solution of potassiumhydrogen sulfate. After warming up to room temperature, the solid wasfiltered and the filtrate concentrated to dryness. The residue wasrepartitioned between 50 mL of ethyl acetate and 25 mL of 1Nhydrochloric acid solution. The layers separated, and the organic layerwas dried over magnesium sulfate, filtered, and concentrated.

Synthesis of (2-Fmoc-amino-3-R′—O-propylamino)-2-substituted acetic acidmethyl ester (10): compounds (10) were prepared by reductive aminationusing sodium cyanoborohydride or sodium triacetoxyborohydride as thereducing agent.

Sodium cyanoborohydride method: To a solution of 8.5 mmol of (2)hydrochloride salt in 20 mL of methanol, kept at room temperature undernitrogen, was added 2.3 mmol of solid potassium hydroxide, and themixture stirred for 25 minutes. A solution of Fmoc-O—R′ serinal (9) in10 mL of methanol was added to the above suspension, and the reactionmixture was stirred for 1 hour. A solution of 8.5 mL of 1N sodiumcyanoborohydride in tetrahydrofuran was added slowly, and the reactionstirred for another 1 hour, filtered, and concentrated. The residue waspartitioned between ethyl acetate and water, and the organic layerwashed with 1×20 mL of saturated sodium bicarbonate, dried over sodiumsulfate, and concentrated.

Sodium triacetoxyborohydride method: A suspension of 21 mmol of (2)hydrochloride salt, and 2.9 mL (21 mmol) of triethyl amine in 50 mL ofdry tetrahydrofuran, was stirred at room temperature for 45 min, andthen a solution of ˜20 mmol crude Fmoc-(O—R′)-serinal (9) in 30 mL oftetrahydrofuran was added, followed by 1.7 g of 4A powdered molecularsieves, and the suspension was stirred for an additional 2 h. 6.4 g (30mmol) of solid sodium triacetoxyborohydride was added, and thesuspension stirred at room temperature overnight. The suspension wasdiluted with methanol, the molecular sieves filtered, and the filtrateconcentrated. The residue was partitioned between 100 mL of ethylacetate and 50 mL of water. The organic layer was dried over sodiumsulfate, filtered, and concentrated.

Compounds (10) were purified by silica gel column chromatography.

R′ R Analytical Data for Compounds (10) ^(t)Bu

¹H NMR δ (CDCl₃): 1.17 (s, 9H, ^(t)Bu), 1.26-1.32 (d, 3H, CH₃), 2.68-2.80 (br. m, 2H, CH₂N), 3.32-3.56 (two br. m, 2H, CH₂O), 3.72 (s, 3H,CH₃O), 3.66-3.82 (m, 1H, CHN), 4.18-4.28 (t, 1H, CH), 4.30-4.46 (d, 2H,CH₂), 5.34-5.44 (br. d, 1H, NH), 7.25-7.44 (two m, 4H, fulvene),7.59-7.64 (d, 2H, fulvene), 7.74-7.79 (d, 2H, fulvene), yield = 57%,t_(R) = 4.93 min, (M⁺ + 1) = 455.67. ^(t)Bu

¹H NMR δ (CDCl₃): 0.88-0.98 (br. t, 6H CH₃), 1.21 (s 9H, ^(t)Bu), 1.26-1.34 (m, 2H, CH₂), 1.44-1.54 (m, 1H, CH), 2.58-2.86 (two m, 1H, CH₂N),3.25-3.35 (m, 1H, CH₂N), 3.37-3.58 (two m, 2H, CH₂O), 3.72- 3.80 (br. m,1H, CHN), 4.14-4.31 (m, 1H, CH), 4.32-4.45 (br. d, 2H, CH₂O), 5.34-5.44(br. d, 1H, NH), 7.30-7.84 (a series of m, 8H, fulvene), yield = 50%,t_(R) = 5.66 min, (M⁺ + 1) = 511.67. ^(t)Bu

¹H NMR δ (CDCl₃): 1.17 (s, 9H, ^(t)Bu), 2.68-2.78 (m, 1H, CH₂N), 2.82-2.92 (m, 1H, CH₂N), 3.35-3.55 (m, 4H, CH₂N, and CH₂O), 3.73 (s, 3H,CH₃O), 3.75-3.85 (m, 1H, CHN), 4.20-4.28 (m, 1H, CH), 4.32-4.48 (m, 2H,CH₂), 5.40-5.50 (d, 1H, NH), 7.28-7.8 (a series of m, 8H, fulvene),yield = 44%, t_(R) = 5.02 min, (M⁺ + 1) = 441.50. ^(t)Bu

¹H NMR δ (CDCl₃): 0.84-0.92 (br. t, 3H, CH₃), 1.17 (s, 9H, ^(t)Bu),1.28- 1.35 (m, 4H, CH₂), 1.48-1.84 (two m, 2H, CH₂), 2.62-2.82 (m, 2H,CH₂N), 3.20-3.33 (m, 1H, CHN), 3.35-3.54 (two m, 2H, CH₂O), 3.72 (s, 3H,CH₃O), 3.64-3.80 (m, 1H, CHN), 4.20-4.28 (t, 1H, CH), 4.32-4.42 (m, 2H,CH₂O), 5.34-5.44 (br. d, 1H, NH), 7.25-7.79 (a series of m, 8H,fulvene), yield = 65%, t_(R) = 5.85 min, (M⁺ + 1) = 441.27. Trt

¹H NMR δ (CDCl₃): 2.36-2.63 (br. m, 2H, CH₂CO), 2.65-2.90 (br. m, 2H,CH₂N), 3.05-3.20 (br. m, 2H, CH₂O), 3.50-3.64 (br. m, 1H, CHN), 3.68 &3.69 (two s, 3H, CH₃O), 3.82-3.94 (br. m, 1H, CHN), 4.12-4.21 (br. m,1H, CH), 4.24-4.43 (br. m, 2H, CH₂O), 4.90-4.98 (br. d, 1H, NH),7.15-7.80 (a series of m, 23H, fulvene and Trt), yield = 39%, t_(R) =8.13 min, (M⁺ + 1) = 926.99. Trt

¹H NMR δ (CDCl₃): 1.68-1.82 (m, 1H, CH₂), 1.85-1.99 (m, 1H, CH₂),2.12-2.37 (m, 2H, CH₂CO), 2.58-2.96 (a series of four m, 2H, CH₂N),3.08-3.28 (br. m, 2H, CH₂O), 3.66 & 3.67 (two s, 3H, CH₃O), 3.76-3.89(br. m, 1H, CHN), 4.15-4.24 (br. m, 1H, CH), 4.28-4.41 (br. d, 2H,CH₂O), 5.10-5.22 (br. d, 1/2H, NH), 5.28-5.35 (br. d, 1/2H, NH), 7.15-7.80 (a series of m, 23H, fulvene, and Trt), yield = 43%, t_(R) = 8.10min, (M⁺ + 1) = 940.97. Trt

¹H NMR δ (CDCl₃): 1.43 (s, 6H, CH₃), 1.46-1.56 (m, 4H, CH₂), 2.06 (s,3H, CH₃), 2.50 (s, 3H, CH₃), 2.57 (s, 3H, CH₃), 2.75-2.80 (m, 1H, CH₂N),2.91 (s, 2H, CH₂), 3.12-3.32 (three br. m, 4H, CH₂N), 3.68 (s, 3H,CH₃O), 4.13-4.21 (t, 1H, CH), 4.28-4.38 (d, 2H, CH₂), 5.12-5.23 (br. d,1H, NH), 5.80-6.12 (two br. m, 2H, NH), 7.18-7.80 (a series of m, 23H,fulvene, and Trt), yield = 68%, t_(R) = 7.52 min, (M⁺ + 1) = 997.91. Trt

¹H NMR δ (CDCl₃): 2.75-2.98 (two m, 2H, CH₂N), 3.06-3.18 (m, 1H, CH₂N),3.22-3.33 (m, 1H, CH₂N), 3.57 & 3.60 (two s, 3H, CH₃O), 3.80-3.92 (m,1H, CHN), 4.00-4.08 (m, 1H, CH), 4.18-4.30 (br. d, 2H, CH₂), 7.00-7.80(a series of m, 25H, fulvene, Trt, and Imidazole), yield = 57%, t_(R) =7.59 min, (M⁺ + 1) = 949.79. Trt

¹H NMR δ (CDCl₃): 1.26 & 1.27 (two s, 9H, ^(t)Bu), 2.50-2.61 (dd, 1H,CH₂-Ph), 2.76-2.86 (m, 2H, CH₂-Ph, and CH₂N), 2.92-3.20 (m, 1H, CH₂N),2.92-3.20 (m, 2H, CH₂O), 3.32-3.46 (m, 1H, CH₂O), 3.59 (s, 3H, CH₂O),3.79-3.88 (m, 1H, CHN), 4.18-4.28 (m, 1H, CH), 4.30-4.37 (br. d, 2H,CH₂O), 5.18-5.26 (br. d, 1H, NH), 6.80-6.88 (d, 2H, Ph), 6.96-7.02 (d,2H, Ph), 7.18-7.80 (a series of m, 23H, fulvene, and Trt), yield = 23%.Trt

¹H NMR δ (CDCl₃): 1.11 (s, 9H, ^(t)Bu), 2.54-2.74 (two m, 2H, CH₂N),3.02-3.58 (six m, 6H, CH₂O, CH₂N, and CHN), 3.70 (s, 3H, CH₃O),3.83-3.93 (m, 1H, CHN), 4.15-4.29 (m, 1H, CH), 4.34-4.37 (d, 2H, CH₂),5.46-5.53 (br. d, 1H, NH), 7.18-7.79 (a series of m, 23H, fulvene, andTrt), yield = 45%, (M⁺ + 1) = 713.42. ^(t)Trt

¹H NMR δ (CDCl₃): 0.80-0.92 (m, 7H, CH₃), 1.75-1.90 (br. m, 1H, CH),2.6-4.36 (a series of m, 9H, CH₂O, CH₂N, CHN), 3.68 (s, 3H, CH₃O), 5.5(d, 0.5H, CH), 7.23-7.77 (m, 24H, fulvene and Trt), yield = 72% (3steps), t_(R) = 6.86 min, (M⁺ + 1) = 669.10.

Synthesis of 4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-one (6):For the preparation of compounds (6) three steps were required: (a) Fmocdeprotection with concomitant cyclization, (b) Fmoc protection, and (c)hydroxyl group deprotection.

Fmoc group removal and cyclization: A solution of 10 mmol of cycliccompound in 30 mL of 30% diethyl amine in ethyl acetate solution wasstirred at room temperature overnight, and then concentrated to dryness.

(a) Fmoc protection: To a biphasic solution of 10 mmol of compound in 20mL of tetrahydrofuran and 10 mL of water, was added 2.52 g (30 mmol) ofsolid sodium bicarbonate, followed by 3.36 g (13 mmol) of Fmoc-Cl. Themixture was stirred for 3 hours, diluted with ethyl acetate, the layersseparated, and the organic layer washed with water, dried over magnesiumsulfate, and concentrated.

(b) Hydroxyl group deprotection: For compounds containing a ^(t)Bu etherprotecting group: The compounds were deprotected with a solution of 90%trifluoroacetic acid in dichloromethane for 1-2 hours, and thenconcentrated to dryness. The residue was dissolved in ethyl acetate andwashed with a saturated solution of sodium bicarbonate, dried overmagnesium sulfate, and then concentrated. For compounds containing a Trtether protecting group: the compounds were deprotected by adding asolution of 1-10% trifluoroacetic acid in dichloromethane containing2-10% tri-isopropyl silane. The reaction was instantaneous. The solutionwas then neutralized by pouring it into a saturated solution of sodiumbicarbonate. The layers were separated, dried over sodium sulfate, andconcentrated.

Compounds (6) were purified by silica gel column chromatography.

R Analytical Data for Compounds (6)

¹H NMR δ (CDCl₃): 1.17-1.35 (b. m, 3 H, CH₃), 2.64-2.82 (t, 1 H, CH₂N),3.2-3.8 (two br. m, 3 H, CH₂O, CH₂N), 4.18-4.44 (br. t, 1 H, CH),4.64-4.90 (br. d, 2 H, CH₂O), 6.70-6.86 (br. s, 1 H, NH), 7.22-7.82 (aseries of m, 8 H, fulvene), yield = 72%, t_(R) = 4.64 min, (M⁺ + 1) =367.32.

¹H NMR δ (CDCl₃): 0.64-1.02 (m, 6 H, CH₃), 1.45-1.63 (m, 3 H, CH₂, andCH), 2.65-2.84 (m, 1 H, CH₂N), 2.89-3.76 (a series of br. m, 5 H, CH₂O,and CHN), 4.17-4.28 (br. m, 1 H, CH), 4.48-4.82 (three br. m, CH₂O, NH,and OH), 6.95- 7.85 (a series of br. m, 8 H, fulvene), yield = 51%,t_(R) = 5.43 min, (M⁺ + 1) = 409.08.

¹H NMR δ (CDCl₃): 3.17-3.78 (a series of br. m, 5 H, CH₂O, CH₂N, andCHN), 421-4.27 (t, 1 H, CH), 4.42-4.68 (br. peak, 2 H, CH₂O), 6.62 (br.s, 1 H, NH), 7.28- 7.81 (a series of m, 8 H, fulvene), yield = 67%,t_(R) = 4.50 min, (M⁺ + 1) = 353.45.

¹H NMR δ (CDCl₃): 0.72-0.90 (br. peak, 3 H, CH₃), 1.0-1.40 (br. peak, 4H, CH₂), 1.48-1.90 (three br. peaks, 2 H, CH₂), 2.68-2.80 (t, 1 H,CH₂N), 3.10-3.70 (four br. peaks, 4 H, CH₂O, CHN, and CH₂N), 4.15-4.25(br. peak, 1 H, CH), 4.54-4.62 (br. d, 2 H, CH₂O), 7.25-7.80 (a seriesof m, 8 H, fulvene), yield = 72%, t_(R) = 5.77 min, (M⁺ + 1) = 408.95.

¹H NMR δ (CDCl₃): 2.50-3.38 (four overlapping br. m, 7 H, CH₂—CO, CH₂N,CH₂O, and CHN), 3.42-3.64 (m, 1/2 H, CHN), 3.70-3.88 (m, 1/2 H, CHN),4.16- 4.23 (br. d, 1 H, CH), 4.48-4.68 (br. m, 2 H, CH₂O), 4.94-5.05(br. d, 1 H, NH), 6.95-7.80 (a series of m, 23 H, fulvene and Trt),yield = 83%, t_(R) = 7.04 min, (M⁺ + 1) = 652.61.

¹H NMR δ (CDCl₃): 1.67-1.78 (br. m, 1 H, CH₂), 1.81-2.0 (br. m, 1 H,CH₂), 2.10- 2.43 (br. m, 2 H, CH₂—CO), 2.58-2.81 (br. m, 2 H, CH₂N),3.02-3.66 (a series of br. m, 4 H, CH₂O, and CHN), 4.17-4.23 (br. m, 1H, CH), 4.40-4.80 (br. m, 2 H, CH₂O), 7.15-7.80 (a series of m, 23 H,fulvene, and Trt), yield = 80%, t_(R) = 7.04 min, (M⁺ + 1) = 666.66.

¹H NMR δ (CDCl₃): 1.43 (s, 6 H, CH₃), 1.50-1.60 (br. m, 4 H, CH₂), 2.10(s, 3 H, CH₃), 2.48 (s, 3 H, CH₃), 2.55 (s, 3 H, CH₃), 2.92 (s, 2 H,CH₂), 3.08-3.47 (two m, 3 H, CH₂O, and CH₂N), 3.57-3.97 (a series of m,3 H, CH₂O, and CHN), 4.15-4.25 (br. m, 1 H, CH), 4.44-4.74 (br. m, 2 H,CH₂), 7.20-7.80 (a series of br. m, 8 H, fulvene), yield = 91%, t_(R) =6.05 min, (M⁺ + 1) = 704.71.

¹H NMR δ (CDCl₃): 2.14-2.56 (two m, 2 H, CH₂-Im), 2.90-3.90 (a series ofm, 4 H, CH₂N, and CH₂O), 4.0-4.74 (a series of m, 4 H, CHN, CH, CH₂),7.0-7.80 (a series of multiples, 25 H, fulvene, Im, and Trt), yield =64%, t_(R) = 5.27 min, (M⁺ + 1) = 675.08.

¹H NMR δ (CDCl₃): 1.29 (s, 9 H, ^(t)Bu) 2.47-2.74 (a series of m, 2 H,CH₂Ph), 2.90-3.04 (m, 1 H, CH₂Ph), 3.06-3.45 (three m, 6 H, CH₂O, andCH₂N), 3.89- 4.29 (three m, 2 H, CH, and CHN), 4.32-4.42 (m, 1 H, CHN),4.56-4.66 (m, 2 H, CH₂), 6.81-7.80 (a series of m, 12 H, fulvene, andPh), yield = 71%, (M⁺ + 1) = 515.81.

¹H NMR δ (CDCl₃): 1.00 & 1.10 (two s, 9 H, ^(t)Bu), 3.0-3.74 (four br.m, 7 H, CH₂O, CH₂N, and CHN), 3.86-4.26 (a series of m, 2 H, CHN, andCH), 4.42-4.68 (br. d, 2 H, CH₂), 7.26-7.80 (a series of br. m, 8 H,fulvene), yield = 55%, (M⁺ + 1) = 439.08.

Synthesis of 4-Fmoc-5-substituted-6-oxo-piperazine-2-carboxylic acid(7): Compounds (7) were prepared as described in method A. Compounds (7)were purified by silica gel column chromatography.

R Analytical Data for Compounds (7)

¹H NMR δ (CDCl₃): 1.08-1.20 (br. peak, 1.5 H, CH₃), 1.30-1.38 (br. peak,1.5 H, CH₃), 2.86-3.07 (br. m, 1 H, CH₂N), 3.83-3.97 (br. m, 1 H, CH₂N),4.18-4.37 (a series of br. peaks, 2 H, CH and CHN), 4.40-4.74 (two br.peaks, 3 H, CH₂O, and CHN), 7.28-7.82 (a series of m, 8 H, fulvene),8.92-9.10 (br. s, 1 H, CO₂H), yield = 51%, t_(R) = 4.80 min,(M⁺ + 1)=381.57.

¹H NMR δ (CDCl₃): 0.40-1.60 (a series of br. peaks, 9 H, CH, CH₂, andCH₃), 2.81-3.09 (br. peak, 1 H, CH₂N), 3.68-3.80 (br. peak, 2 H, CHN),3.96-4.32 (br. peaks, 2 H, CH, and CNH), 4.48-4.68 (br. peak, CH₂O),7.26-7.84 (a series of m, 8 H, fulvene), yield = 50%, t_(R) = 5.57 min,(M⁺+ 1), = 423.15.

¹H NMR δ (CDCl₃): 3.77-3.99 (m, 1 H, CHN), 3.90-4.35 (a series of m, 5H, CH₂N, CH), 4.44-4.57 (d, 2 H, CH₂), 7.3-7.82 (a series of m, 8 H,fulvene), yield = 48%, t_(R) = 4.58 min, (M⁺ + 1) = 367.30.

¹H NMR δ (CDCl₃): 0.69-1.90 (a series of br. peaks, CH₂, and CH₃),2.85-3.05 (br. peak, 2 H, CH₂N), 3.65-3.95 (two br. peaks, 1 H, CHN),4.00-4.40 (two br. peaks, CH₂N, and CH), 4.41-4.74 (br. peak, 3 H, CH₂O,and CHN), 7.20-7.80 (a series of br. m, 8 H, fulvene), yield = 70%,t_(R) = 5.93 min, (M⁺ + 1) = 423.42.

¹H NMR δ (CDCl₃): 2.51-3.06 (a series of m, 2 H, CH₂—CO), 3.85-4.86 (aseries of m, 7 H, CH₂N, CHN, CH, and CH₂O), 7.0-7.78 (a series of br. m,23 H, fulvene and Trt), yield = 30%, t_(R) = 7.04 min, (M⁺ + 1) =666.79.

¹H NMR δ (CDCl₃): 1.74-2.46 (a series of br. m, 4 H, CH₂—CO, and CH₂),3.78- 4.06 (two m, 2 H, CH₂N), 4.16-4.68 (a series of br. m, 5 H, CHN,CH, and CH₂O), 7.14-7.82 (a series of br. m, 23 H, fulvene, and Trt),yield = 47%, t_(R) = 7.11 min, (M⁺ + 1) = 680.33.

¹H NMR δ (CDCl₃): 1.08-1.60 (a series of br. peaks, 8 H, CH₂, and CH₃),2.12 (s, 3 H, CH₃), 2.48 (s, 3 H, CH₃), 2.57 (s, 3 H, CH₃), 2.92 (s, 2H, CH₃), 3.10-3.25 (br. m, 2 H, CH₂N), 3.82-4.28 (a series of br. m, 4H, CH₂N, CHN, CH), 4.40- 4.70 (br. m, 3 H, CHN, and CH₂O), 7.20-7.80 (aseries of br. m, 8 H, fulvene), yield = 42%,t_(R) = 6.15 min, (M⁺ + 1) =718.69.

¹H NMR δ (CDCl₃): 1.28 & 1.34 (two s, 9 H, ^(t)Bu), 2.42-3.64 (a seriesof br. m, 5 H, CH₂N, CHN, and CH₂Ph), 4.0-4.76 (a series of br. m, 4 H,CHN, CH, and CH₂O), 6.60-6.96 (br. m, 4 H, Ph), 7.20-7.80 (a series ofbr. m, 8 H, fulvene), yield = 67%, (M⁺ + 1) = 529.17.

¹H NMR δ (CDCl₃): 0.96- & 1.10 (two s, 9 H, ^(t)Bu), 3.04-3.18 (br. m,0.5 H, CH₂), 3.30-3.94 (four br. m, 3.5 H, CH₂N, and CH₂O), 3.98-4.32(br. m, 2 H, CH, and CHN), 4.33-4.74 (two br. m, 3 H, CHN, CH₂O),7.28-7.80 (a series of m, 8 H, fulvene), yield = 60%, (M⁺ + 1) = 453.37.

Method E: (2-Fmoc-amino-3-hydroxy-propyl-Cbz-amino)-2-substituted aceticacid methyl ester (15) were prepared by reductive amination of Fmocserinal (OR′) (9) with an α amino ester (2), using either sodiumcyanoborohydride or sodium triacetoxyborohydride as the reducing agent.The secondary amine was protected with benzylchloroformate, and then thehydroxyl group deprotected with trifluoroacetic acid solution. Compounds(15) were then Fmoc deprotected. The amino ester intermediates cyclizedimmediately to form 4-Cbz-3-substituted 6-hydroxymethyl-piperazin-2-ones(16). Fmoc 3-substituted 6-hydroxymethyl-piperazin-2-ones (6) wereprepared by protecting group exchange, and then oxidized to the desiredproducts (7) as described in method A.

Synthesis of (2-Fmoc-amino-3-hydroxy-propyl-Cbz-amino)-2-substitutedacetic acid methyl ester (15): A suspension of 67 mmol of amino esterhydrochloride (2), and 20.9 mmol of solid potassium hydroxide in 80 mLof methanol was stirred at room temperature for 25 minutes, and thenadded to a suspension of (9) in 250 mL of methanol. The reaction mixturewas stirred for 1.5 hours, followed by the slow addition of 70 mL of 1Nsodium cyanoborohydride solution in tetrahydrofuran. The reaction wasstirred overnight, and then concentrated. The residue was partitionedbetween 300 mL of tetrahydrofuran and 50 mL of 1N hydrochloric acidsolution. The layers were separated, and the organic layer neutralizedwith a solution of 239 mmol of sodium bicarbonate in 50 mL of water, andthen 66 mmol of benzyl chloroformate was added slowly, and the reactionwas stirred for 3 hours, diluted with 200 mL of ethyl acetate, and thelayers separated. The organic layer was dried over magnesium sulfate,and concentrated. The residue was dissolved in a solution oftrifluoroacetic acid in dichloromethane, and stirred at room temperaturefor 2 hours. The solution was poured over 200 mL of saturated sodiumbicarbonate solution. The layers separated, and the organic layer wasdried over magnesium sulfate, and concentrated. Compounds (15) werepurified by silica gel column chromatography.

R Analytical Data for Compounds (15)

¹H NMR δ (CDCl₃): 1.38-1.45 (d, 9 H, ^(t)Bu), 2.68-2.78 (m, 1/2 H,CH₂—CO), 3.0-3.20 (m, and s together, 3.5 H, CH₂—CO, CH₂—O, and CH₃—O),3.52-3.60 (m, 1 H, CH₃—O), 3.96-4.40 (a series of multiples, 4 H),4.96-5.10 (m, 2 H, CH₂—O), 5.77-5.83 (m, 1/2 H, NH), 7.14-7.79, (aseries of m, 23 H, Trt and fulvene), yield = 70%, t_(R) = 9.82 min.

Synthesis of 4-Cbz-6-hydroxymethyl-3-substituted-piperazin-2-ones (16):A solution of 24 mmol of (15) in 100 mL of 30% diethyl amine in ethylacetate was stirred at room temperature overnight, and then concentratedto dryness. The compounds were purified by silica gel columnchromatography.

R Analytical Data for Compounds (16)

¹H NMR δ (CDCl₃): 1.36 (d, 9 H, ^(t)Bu), 2.60-2.90 (m, 2 H, CH₂—CO),2.94-3.20 (br. m, 2 H, CH₂N, 3.38-3.50 (br. m, 2 H, CH₂—O), 3.86-4.20(m, 1 H, CH—N), 4.74-4.84 (br, 1 H, OH), 5.10-5.15 (s, 2 H, CH₂—O),7.26-7.36 (s, 5 H, Ph), 7.87-7.95 (s, 1 H, NH), yield = 70%, t_(R) =4.66 min, (M⁺ + 1) = 379.41.

Synthesis of 4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-ones (6):A suspension of 15 mmol of (16), and 1.8 g of 10% palladium on carbon in50 mL of ethanol was hydrogenated at room temperature and atmosphericpressure until HPLC showed that the reaction was complete. The mixturewas then filtered through celite, concentrated, and the residue wasdissolved in 35 mL of tetrahydrofuran, and 10 mL of water, and then 62mmol of solid sodium bicarbonate was added, followed by 16 mmol ofFmoc-Cl, and the mixture was stirred for 3 hours, diluted with 100 mL ofethyl acetate and 10 mL of water. The layers were separated, and theorganic layer dried over magnesium sulfate, and concentrated. Compounds(6) were purified by silica gel column chromatography.

R Analytical Data for Compounds (6)

¹H NMR δ (CDCl₃): 1.41 (s, 9 H, ^(t)Bu), 2.20-2.40 (m, 1/2 H, CH₂—CO,2.64-2.96 (m, 1.5 H, CH₂—CO), 2.98-3.16 (m, 1 H, CH₂O), 3.2-3.8 (aseries of br. m, 4 H, CH₂O, and CH₂N), 4.20-4.38 (two m, CHN, and CH),4.5-4.67 (br. m, 2 H, CH₂O), 4.70- 4.83 (br. m, 1/2 H, NH), 7.27-7.84 (aseries of m, 8 H, fulvene), yield = 77%, t_(R) = 5.78 min, (M⁺ + 1) =467.82.

Synthesis of 4-Fmoc-5-substituted-6-oxo-piperazine-2-carboxylic acid(7): Compounds (7) were prepared as described in method A, and purifiedby silica gel column chromatography.

R Analytical Data for Compounds (7)

¹H NMR δ (CDCl₃): 1.4 (s, 9 H, ^(t)Bu), 2.20-2.33 (br. d, 1 H, CH₂—CO),2.55-2.67 (br. d, 1 H, CH₂—CO), 3.25-3.52 (br. m, 2 H, CH₂N), 3.82-3.94,and 4.07-4.18 (br. peaks, 1 H, CHN), 4.20-4.42 (m, 2 H, CHN, CH),4.50-4.72 (m, 2 H, CH₂—O), 7.30-7.82 (8 H, fulvene), 9.20-9.35 (br. s, 1H, CO₂H), yield = 63%, t_(R) = 6.60 min,(M⁺ + 1) = 481.17.

Method F: (2-Cbz-amino-3-benzyloxy-propylamino)-2-substituted aceticacid methyl esters (20) were prepared by reductive amination of Cbzserinal (OBn) (19) with an α-amino ester (2), using either sodiumcyanoborohydride or sodium triacetoxyborohydride as the reducing agent.The Cbz O-Benzyl serinal (19) required for the reductive amination wasobtained by oxidation of Cbz serinol (OBn) (18) with Dess-Martinperiodinane. Hydrogenation of (20) followed by cyclization gave3-substituted 6-hydroxymethyl-piperazin-2-ones which was then Fmocprotected to 4-Fmoc-3-substituted 6-hydroxymethyl-piperazin-2-ones (6).The final products (7) were obtained as described in method A.

Synthesis of Cbz-serinol (OBn) (18): Compound (18) was prepared asdescribed for compound (13). Compound (18) was obtained in 79% yieldafter silica gel column chromatography purification. ¹H NMR δ (CDCl₃)3.57-3.74 (two m, 3H, CHN, and CH₂O), 3.76-3.96 (two m, 2H, CH₂O), 4.50(s, 2H, CH₂O), 5.10 (s, 2H, CH₂O), 5.40-5.50 (br. d, 1H, NH), 7.22-7.38(m, 10H, Ph); HPLC t_(R)=5.33 min, (M++Na⁺)=337.64.

Synthesis of Cbz serinal (OBn) (19): Compound (19) was prepared asdescribed for compound (9). To a solution of 80 mmol of Cbz-O-Bn serinol(18) in 200 mL of dry dichloromethane, kept at room temperature undernitrogen, was added 88 mmol of Dess-Martin periodinane, and the reactionstirred for 2.5 hours, and then quenched by addition of 400 mL of 10%aqueous sodium thiosulfate solution. The layers were separated, and theorganic layer concentrated, diluted with 300 mL of ethyl ether, andwashed three times with a saturated aqueous bicarbonate solutioncontaining 10% sodium thiosulfate, dried over magnesium sulfate, andconcentrated. Compound (19) was obtained in 99% crude yield, and usedwithout further purification. ¹H NMR δ (CDCl₃) 3.69-3.78 (dd, 1H, CH₂O),3.99-4.06 (dd, 1H, CH₂O), 4.37-4.46 (m, 1H, CHN), 4.47-4.52 (d, 2H,CH₂O), 5.14 (s, 2H, CH₂O), 5.65-5.75 (br. d, 1H, NH), 7.14-7.48 (aseries of m, 9H, Ph), 7.98-8.08 (dd, 1H, Ph), 9.63 (s, 1H, CHO).

Synthesis of (2-Cbz-amino-3-benzyloxy-propylamino)-2-substituted aceticacid methyl esters (20): Compounds (20) were prepared as described forcompound (10), but using Cbz serinal (19) as the aldehyde. Compounds(20) were purified by silica gel column chromatography.

R Analytical Data for Compounds (20)

¹H NMR δ (CDCl₃): 1.30 (s, 9 H, ^(t)Bu), 2.50-2.96 (m, 3 H, CH₂Ph, andCH₂N), 3.28-3.54 (m, 3 H, CH₂N, and CH₂O), 3.59 and 3.61 (two s, 3 H,CH₃O), 3.68-3.86 (m, 1 H, CHN), 4.41-4.45 (d, 2 H, CH₂O), 5.08 (s, 2 H,CH₂O), 5.25-5.37 (br. t, 1 H, NH), 6.84-6.88 (d, 2 H, Ph), 6.98-7.04 (d,2 H, Ph), 7.24-7.37 (m, 10 H, Ph), yield = 50%,(M⁺ + 1) = 549.35.

Synthesis of 4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-ones (6):A suspension of 38 mmol of (20) in 160 mL of ethanol, 38 mL of 1Nhydrochloric acid, and 20 g of 10% palladium on carbon was hydrogenatedat room temperature and atmospheric pressure until HPLC showed that thereaction was complete. The mixture was then filtered through celite, andconcentrated to dryness. The residue was diluted with 75 mL oftetrahydrofuran and neutralized with a saturated sodium bicarbonatesolution. 106 mmol of solid sodium bicarbonate, and 53 mmol of Fmocchloride were added, and the reaction stirred at room temperature untilHPLC showed the reaction was complete, diluted with 300 mL of ethylacetate and 300 mL of brine. The layers were separated, and the organiclayer washed twice with brine, dried over magnesium sulfate, andconcentrated. The products (6) were purified by silica gel columnchromatography.

Synthesis of 4-Fmoc-5-substituted-6-oxo-piperazine-2-carboxylic acid(7): Compounds (7) were prepared as described in method A.

Synthesis of 2,2-disubstituted Ketopiperazine Scaffolds Mimicking AminoAcids Without Functionalized Side Chains (Method G)

The syntheses of4-Fmoc-5-substituted-6-oxo-piperazine-2-methyl-2-carboxylic acidscaffolds mimicking amino acids without functionalized side chains wascarried out using method G.2-Boc-amino-3-methoxycarbonyl-1-substituted-methylamino-2-methyl-propionicacid tert-butyl esters (23) were prepared by reductive amination of2-Boc-amino-2-methyl-3-oxo-propionic acid methyl ester (22) with anα-amino ester (2), using either sodium cyanoborohydride or sodiumtriacetoxyborohydride as the reducing agent. Compound (22) required forthe reductive amination was obtained by oxidation of α-methyl-Boc serinetert-butyl ester (21) with Dess-Martin periodinane. The Boc group of(23) was removed with 2N hydrogen chloride in dioxane, and the aminoesters cyclized to unprotected5-substituted-6-oxo-piperazine-2-methyl-2-carboxylic acid tert-butylesters (24), which were protected with Fmoc chloride to give4-Fmoc-5-substituted-6-oxo-piperazine-2-methyl-2-carboxylic acidtert-butyl esters, which were deprotected with trifluoroacetic acid togive the final products (25).

Synthesis of 2-Boc-amino-2-methyl-3-oxo-propionic acid tert-butyl ester(22): Oxidation of Boc α-Methyl serine tert-butyl ester (21) was doneusing Dess-Martin periodinane as describe before gave the desiredproduct (22) in 96% crude yield. The compound was used without furtherpurification in the next step. ¹H NMR δ (CDCl₃): 1.44 (s, 18H, ^(t)Bu),1.46 (s, 3H, CH₃), 5.63-5.70 (br. s, 1H, NH), 9.5 (s, 1H, CHO)

Synthesis of2-Boc-amino-3-methoxycarbonyl-1-substituted-methylamino-2-methyl-propionicacid tert-butyl ester (23): Compounds (23) were prepared using aprocedure similar to the one described for compound (10), but usingcompound (22) as the aldehyde. Compounds (23) were purified by silicagel column chromatography.

R Analytical Data for Compounds (23)

¹H NMR δ (CDCl₃): 1.40-1.46 (two s, 21 H, CH₃ and ^(t)Bu), 2.60-2.72(br. m, 1 H, CH₂Ph), 2.82-3.00 (m, 3 H, CH₂Ph, and CH₂N), 3.32-3.43 (t,1 H, CHN), 3.65 (s, 3 H, CH₃), 5.62 (br. s, 1 H, NH), 7.13-7.32 (m, 5 H,Ph), yield = 69%, (M⁺ + 1) = 436.98.

Synthesis of 2-methyl-6-oxo-5-substituted-piperazine-2-carboxylic acid(25): A solution of 4 mmol of (23) in 8 mL of 2N hydrogen chloride indioxane was stirred at room temperature for 5 hours, and thenconcentrated to dryness. The residue was suspended in 20 mL oftetrahydrofuran, neutralized with 10 mmol of triethylamine, and stirredat 60° C. for 2 days. It was then concentrated to dryness, resuspendedin 20 mL of tetrahydrofuran and 10 mL of water, solid sodium bicarbonatewas added to adjust the pH to basic, followed by 5.6 mmol of solid Fmocchloride, and the reaction mixture stirred overnight at roomtemperature, the pH adjusted to 1 with 1N hydrochloric acid solution,diluted with 100 mL of ethyl acetate, and the layers separated. Theorganic layer was washed with 2×100 mL of brine, dried over magnesiumsulfate and concentrated. The residue was dissolved in 10 mL of 50%trifluoroacetic acid in dichloromethane, and the solution stirred atroom temperature for 3 hours. The solvent was concentrated, and theproducts (25) purified by silica gel column chromatography.

R Analytical Data for Compounds (25)

¹H NMR δ (CDCl₃): 1.12 (s, 3 H, CH₃), 2.50-2.62 (m, 0.5 H, CH₂Ph),2.96-3.38 (three m, 1.5 H, CH₂Ph), 3.86-4.52 (a series of m, 6 H, CHN,CH, and CH₂O), 6.80-7.80 (a series of m, 13 H, fulvene and Ph), yield =22%, (M⁺+ 1) = 471.47Synthesis of 2,2-disubstituted Ketopiperazine Scaffolds Mimicking AminoAcids with Functionalized Side Chains (Method H)

The syntheses of4-Fmoc-5-substituted-6-oxo-piperazine-2-methyl-2-carboxylic acidscaffolds mimicking amino acids with functionalized side chains areperformed using method H.2-Alloc-amino-3-methoxycarbonyl-1-substituted-methylamino-2-methyl-propionicacid methyl ester (30) is prepared by reductive amination of2-Alloc-amino-2-methyl-3-oxo-propionic acid methyl ester (28) with anα-amino allyl ester (29), using either sodium cyanoborohydride or sodiumtriacetoxyborohydride as the reducing agent, followed by protection ofthe secondary amine with benzylchloroformate. Compound (28) required forthe reductive amination is obtained by oxidation of (27) withDess-Martin periodinane. The allyl ester and the alloc groups of analogs(30) are removed using tetrakistriphenyl phosphine palladium (0) and theamino acid cyclized by reaction with a peptide coupling reagent to give5-substituted-6-oxo-piperazine-2-methyl-2-carboxylic acid methyl esters(31). 4-Fmoc-5-substituted-6-oxo-piperazine-2-methyl-2-carboxylic acids(25) are obtained by saponification of the methyl ester, followed byprotecting group exchange.

Synthesis of Alloc α-methyl serine methyl ester (27): A solution of 8mmol of Boc α-methyl serine (26), 1.0 g (12 mmol) of solid sodiumbicarbonate, and 1.0 mL (16 mmol) of iodomethane in 8 mL of drydimethylformamide, kept under nitrogen is stirred overnight. Thereaction mixture is then poured over 50 mL of water, and extracted with50 mL of diethyl ether, and washed with 1×20 mL of water, dried overmagnesium sulfate, and concentrated. The residue is dissolved in 20 mLof 90% trifluoroacetic acid in dichloromethane, and the solution isstirred at room temperature for 3 hours, and then concentrated todryness. The residue is dissolved in 35 mL of tetrahydrofuran, and 10 mlof water, followed by addition of 30 mmol of solid sodium bicarbonate,and the slow addition of 12 mmol of allyl chloroformate. The mixture isstirred at room temperature for 2 hours, diluted with 50 mL of ethylacetate, and the layers separated. The organic layer is then washed with1×10 mL of saturated sodium bicarbonate, and 1×10 ml of 1N hydrochloricacid, and 1×10 mL of water, dry over magnesium sulfate, andconcentrated. Compound (27) is purified by silica gel columnchromatography.

Synthesis of 2-Alloc-amino-2-methyl-3-oxo-propionic acid methyl ester(28): Oxidation of Alloc α-methyl serine methyl ester (27) is done usingDess-Martin periodinane as described above to yield the desired product(28).

Synthesis of2-Alloc-amino-3-methoxycarbonyl-1-substituted-methyl-Cbz-amino-2-methyl-propionicacid allyl ester (30): Compounds (30) are prepared using a proceduresimilar to the one described for compounds (15), but using compound (28)as the aldehyde.

Synthesis of 4-Cbz-2-methyl-6-oxo-5-substituted-piperazine-2-carboxylicacid methyl ester (31): To solution of 10 mmol of compound (30) in 30 mLof dichloromethane, kept at room temperature under nitrogen, is added 2equivalents of phenylsilane and 0.3 equivalents oftetrakistriphenylphosphine palladium (0), and the solution stirred for 2hours, and then 11 mmol of TBTU, and 14 mmol of N-methyl-morpholine areadded, and the solution stirred at room temperature for 2 hours, andthen concentrated to dryness.

Synthesis of 4-Fmoc-2-methyl-6-oxo-5-substituted-piperazine-2-carboxylicacid (25): To a solution of 10 mmol of compound (31) in 25 mL ofmethanol, kept at room temperature under nitrogen, is added slowly 11mmol of 1N sodium hydroxide solution, and the reaction is stirred atroom temperature overnight, neutralized with 21 mL of 1N hydrochloricacid solution, 1 g of 10% palladium on carbon is added, and thesuspension hydrogenated at room temperature and atmospheric pressure for3 hours. The suspension is filtered through celite and concentrated. Theresidue is redissolved in 25 mL of tetrahydrofuran, and 10 mL of water,followed by the addition of 30 mmol of solid sodium bicarbonate, and 10mmol of Fmoc chloride, and the reaction is stirred at room temperatureunder nitrogen for 2 hours. The reaction is then diluted with 50 mL ofethyl acetate, and acidified with 1N hydrochloric acid solution. Thelayers are then separated, and the organic layer is washed with 1×20 mLof water, dried over magnesium sulfate, and concentrated. Compounds (25)are purified by silica gel column chromatography.

Synthesis of (5-substituted-6-oxo-piperazin-2-yl)-acetic acid Scaffolds(Methods I, J, K)The syntheses of (5-substituted-6-oxo-piperazin-2-yl)-acetic acidscaffolds were carried out by several methods.

Method I: (tert-butyl3-protected-amino-4-(methoxycarbonyl-substituted-methylamino)-butyrates(35) were prepared by reductive amination of tert-butyl3-protected-amino-4-oxo-butyrate (34) with α-amino esters (2), usingeither sodium cyanoborohydride or sodium triacetoxyborohydride as thereducing agent. The tert-butyl 3-protected-amino-4-oxo-butyrate (34)required for the reductive amination was prepared by lithium aluminumhydride (LAH) reduction of the Weinreb amide derivatives (33).Tert-butyl(3-protected-amino-4-(methoxycarbonyl-substituted-methylamino)-butyrateanalogs (35) were then deprotected, cyclized, and Fmoc protected to givetert-butyl (5-substituted-6-oxo-piperazin-2-yl)-acetates (36), whichwere then deprotected to give the final products (37).

Synthesis of amino protected Asp-(O^(t)Bu) Weinreb amide (33): Compounds(33) were prepared using a procedure similar to the one described forcompound (14).

R₂ Analytical Data for Compounds (33) Cbz ¹H NMR δ (CDCl₃): 1.40 (s, 9H,^(t)Bu), 2.47-2.59 (dd, 1H, CH₂CO), 3.20 (s, 3H, CH₂N), 3.77 (s, 3H,CH₃O), 4.96-5.05 (br. m, 1H, CHN), 5.05-5.12 (br. d, 2H, CH₂O),5.58-5.66 (br. d, 1H, NH), 7.30-7.36 (br. m, 5H, Ph), yield = 90% Fmoc¹H NMR δ (CDCl₃): 1.45 (s, 9H, ^(t)Bu), 2.55-2.64 (dd, 1H, CH₂CO),2.69-2.80 (dd, 1H, CH₂O), 3.60 (s, 3H, CH₃N), 3.79 (s, 3H, CH₃O),4.18-4.26 (t, 1H, CH), 4.32- 4.40 (d, 2H, CH₂O), 4.98-5.19 (m, 1H, CHN),5.70-5.76 (br. d, 1H, NH), 7.35-7.80 (a series of m, 8H, fulvene), yield= quant.

Synthesis of tert-butyl 3-amino protected-amino-4-oxo-butyrate (34):Compounds (34) were prepared using a procedure similar to the onedescribed for compound (9).

R₂ Analytical Data for Compounds (34) Cbz ¹H NMR δ (CDCl₃): 1.40 (s, 9H,^(t)Bu), 2.69-2.81 (dd, 1H, CH₂CO), 2.89-3.01 (dd, 1H, CH₂CO), 4.33-4.42(m 1H, CHN), 5.12 (s, 2H, CH₂O), 5.83-5.88 (br. d, 1H, NH), 7.31-7.39(br. m, 5H, Ph), 9.64 (s, 1H, CHO) Fmoc ¹H NMR δ (CDCl₃): 1.45 (s, 9H,^(t)Bu), 2.58-3.02 (a series of m, 2H, CH₂CO), 4.20- 4.28 (t, 1H, CH),4.35-4.49 (m, 3H, CH₂O, and CHN), 5.85-5.92 (br. d, 1H, NH), 7.27-7.80(a series of m, 8H, fulvene), 9.65 (s, 1H, CHO)

Synthesis of tert-butyl3-Protected-amino-4-(methoxycarbonyl-substituted-methylamino)-butyrate(35): Compounds (35) were prepared using a procedure similar to the onedescribed for compounds (10), but using compounds (34) as the aldehyde.

R₂ R Analytical Data for Compounds (35) Cbz

¹H NMR δ (CDCl₃): 1.40 (s, 9 H, ^(t)Bu), 2.27-3.02 (a series of m, 6 H,CH₂CO, CH₂Ph, and CH₂N), 3.43-3.52 (t, 1 H, CHN), 3.65 (s, 3 H, CH₃O),3.84-3.98 (m, 1 H, CHN), 5.08 (s, 2 H, CH₂O), 5.33-5.44 (br. d, 1 H,NH), 7.11-7.42 (a series of m, 10 H, Ph), yield = 60%, t_(R) = 4.79 min,(M⁺ + 1) = 471.20. Cbz

¹H NMR δ (CDCl₃): 1.55 (s, 9 H, ^(t)Bu), 2.42-2.68 (br. m, 2 H, CH₂N),2.74-2.92 (two dd, 2 H, CH₂O), 3.46-3.50 (d, 2 H, CH₂N), 3.78 (s, 3 H,CH₃O), 4.02-4.14 (m, 1 H, CHN), 5.15 (s, 2 H, CH₂O), 7.40-7.45 (m, 5 H,Ph), t_(R) = 3.82, (M⁺ + 1) = 381.28 Cbz

¹H NMR δ (CDCl₃): 1.25-1.30 (d, 3 H, CH₃), 1.44 (s, 9 H, ^(t)Bu), 2.38-2.65 (a series of m, 2 H, CH₂CO), 2.66-2.85 (m, 2 H, CH₂N), 3.60-3.70(m, 1 H, CHN), 3.7 (s, 3 H, CH₃O), 3.9-4.1 (m, 1 H, CHN), 5.1 (s, 2 H,CH₂O), 5.4-5.6 (br. t, 1 H, NH), 7.28-7.4 (m, 5 H, Ph), t_(R) = 3.81min, (M⁺ + 1) = 395.25. Cbz

¹H NMR δ (CDCl₃): 0.84-0.91 (m, 6 H, CH₃), 1.08-1.30 (m, 1 H, CH), 1.45(s, 9 H, ^(t)Bu), 1.45-1.70 (m, 2 H, CH₂), 2.39-2.60 (m, 3 H, CH₂CO,CH₂N), 2.74-2.86 (dd, 1 H, CH₂N), 2.98-3.16 (dd, 1 H, CHN), 3.7 (s, 3 H,CH₃O), 3.92-4.08 (br. m, 1 H, CHN), 5.1 (s, 2 H, CH₂O), 7.26-7.45 (m, 5H, Ph), t_(R) = 4.56 min,(M⁺ + 1) = 437.31.

Synthesis of tert-butyl(4-Fmoc-5-substituted-6-oxo-piperazin-2-yl)-acetate (36): For compoundscontaining Fmoc amino protecting group, a solution of 10 mmol ofcompound (35) in 30 mL of 30% diethyl amine in ethyl acetate solutionwas stirred at room temperature overnight, and then concentrated todryness. For compounds containing Cbz amino protecting group, a solutionof 10 mmol of compound (35) in 30 mL of ethanol was hydrogenated at roomtemperature and atmospheric pressure for 2 hours, filtered throughcelite, and concentrated to dryness. For Fmoc protection, the residuewas dissolved in 20 mL of tetrahydrofuran, and 10 mL of water, and 2.52g (30 mmol) of solid sodium bicarbonate was added, followed by theaddition of 3.3 g (13 mmol) of Fmoc-Cl. The mixture was stirred for 3hours and diluted with ethyl acetate. The layers separated, and theorganic layer was washed with water, dried over magnesium sulfate, andconcentrated. Compounds (36) were purified by silica gel columnchromatography.

R Analytical Data for Compounds (36)

¹H NMR δ (CDCl₃): 1.44 (s, 9 H, ^(t)Bu), 1.71-2.10 (m, 2 H, CH₂CO),2.10-2.30 (br. d, 1 H, CHN), 2.62-2.82 (br. d, 1 H, CH₂Ph), 2.90-3.74 (aseries of br. m, 3 H, CH₂N, CHN), 3.80-4.07 (br. d, 1 H, CHN), 4.10-4.50(br. m, 3 H, CH₂O, and CH), 6.74-7.80 (a series of m, 23 H, fulvene, andPh), yield = 75%, t_(R) = 7.15 min, (M⁺ + 1) = 527.20.

¹H NMR δ (CDCl₃): 0.77-1.94 (a series of m, and two s, 18 H, ^(t)Bu,CH₂, and CH₃), 2.07-2.76 (three m, 3 H, CH₂CO, and CHN), 2.86-3.80 (fourm, 2 H, CH₂N), 4.16-4.27 (m, 1 H, CH), 4.30-4.43 (m, 1 H, CHN),4.50-4.70 (br. m, 2 H, CH₂O), 7.26-7.79 (a series of m, 8 H, fulvene),yield = 40% for three steps, t_(R) = 7.31 min,(M⁺ + 1) = 493.47.

¹H NMR δ (CDCl₃): 1.45 (s, 9 H, ^(t)Bu), 1.9-2.5 (m 2 H, CH₂CO),3.02-4.7 (a series of m, 8 H, CH, CH₂, CH₂N), 7.25-7.78 (three m, 8 H,fulvene), t_(R) = 6.42 min, (M⁺ + 1) = 431.31.

¹H NMR δ (CDCl₃): 1.20-1.35 (br. m, 3 H, CH₃), 1.45 (s, 9 H, ^(t)Bu),2.1-2.80 (three m, 3 H, CH₂CO, CH₂N), 3.1-4.1 (four m, 3 H, CH₂N, CHN),4.18-4.26 (br. t, 1 H, CH), 4.28-4.46 (br. m, 1 H, CHN), 4.50-4.68 (br.m, 2 H, CH₂), 7.28-7.8 (three m, 8 H, fulvene), t_(R) = 6.29 min,(M⁺ + 1) = 451.24.

¹H NMR δ (CDCl₃): 1.20-1.60 (br. m, and s, 15 H, CH₃, ^(t)Bu), 2.21-2.80(3 br. m, 2 H, CH₂CO), 3.0-3.9 (four br. m, 2 H, CH₂N), 4.18-4.26 (br.m, 2 H, CH, CHN), 4.38-4.86 (br. m, 3 H, CH₂, CHN), 7.26-7.86 (a seriesof m, 8 H, fulvene), t_(R) = 6.90 min, (M⁺ + 1) = 493.31.

Synthesis of (4-Fmoc-5-substituted-6-oxo-piperazin-2-yl)-acetate (37):Compounds (36) were deprotected with 90% trifluoroacetic acid solutionin dichloromethane for 3 hours, and then concentrated to dryness. Finalproducts (37) were purified by silica gel column chromatography.

R Analytical Data for Compounds (37)

¹H NMR δ (CDCl₃): 1.82-2.13 (br. t, 1 H, CHN), 2.32-2.53 (br. d, 1 H,CH₂CO). 2.63-2.81 (br. d, 1 H, CH₂CO), 2.90-3.29 (two br. m, CH₂Ph),3.38-3.59 (br. m, 1 H, CH₂N), 3.66-3.85 (br. m, 1 H, CH₂N), 3.95-4.24(two overlapping br. peaks, 2 H, CHN, CH), 4.30-4.93 (br. d, 2 H, CH₂O),6.84-7.82 (a series of m, 13 H, fulvene, and Ph), 8.08-8.25 (br. d, 1H,CO₂H), yield = quant., t_(R) = 5.57 min, (M⁺ + 1) = 471.07.

¹H NMR δ (CDCl₃): 0.72-1.92 (five br. m, 9 H, CH₂, and CH₃) , 2.14-2.70(two br m, 3 H, CH₂CO, and CHN), 3.26-3.62 (two br. m, 1 H, CH₂N),3.70-3.90 (br. m, 1 H, CH₂N), 4.03-4.30 (two m, 2 H, CHN, and CH),4.42-4.82 (br. m, 2 H, CH₂O), 7.28-7.82 (a series of m, 8 H, fulvene),7.97 (s, 1 H, CO₂H), yield = 90%, t_(R) = 5.61 min,(M⁺ + 1) = 437.76.

¹H NMR δ (CDCl₃): 2.10-2.66 (m, 2 H, CH₂CO), 3.2-3.92 (four m, 3 H,CH₂N, CHN), 3.97-4.06 (m, 1 H, CH), 4.2-4.3 (m, 2 H, CH₂), 4.48-4.62 (m,2 H, CH₂N), 7.24-7.81 (a series of m, 8 H, fulvene), t_(R) = 4.74 min,(M⁺ + 1) = 381.13.

¹H NMR δ (CDCl₃): 1.15-1.37 (br. m, 3 H, CH₃), 2.22-2.78 (three br. m, 2H, CH₂CO), 3.0-4.10 (five br. m, 3 H, CH₂N, CHN), 4.15-4.40 (m, 1 H,CH), 4.45-4.7 (br. m, 3 H, CH₂, CHN), 7.26-8.10 (a series of m, 8 H,fulvene), t_(R) = 4.66 min, (M⁺ + 1) = 395.32.

¹H NMR δ (CDCl₃): 0.6-1.2 (m, 6 H, CH₃), 1.22-2.8 (four m, 4 H, CH₂CO,CH₂), 3.1-4.0 (five m, 3 H, CH₂N, CHN), 4.18-4.32 (m, 1 H, CH),4.41-4.84 (m, 3 H, CH₂, CHN), 7.26-8.2 (a series of m, 8 H, fulvene),t_(R) = 5.46 min, (M⁺ + 1) = 437.37.

Method J: Diphenylmethyl3-Fmoc-amino-4-(methoxycarbonyl-substituted-methylamino)-butyrates (41)were prepared by reductive amination of diphenylmethyl3-Fmoc-amino-4-oxo-butyrate (40) with α-amino esters (2), using eithersodium cyanoborohydride or sodium triacetoxyborohydride as the reducingagent. The diphenylmethyl 3-Fmoc-amino-4-oxo-butyrate (40) required forthe reductive amination was prepared by lithium aluminum hydridereduction of the Weinreb amide derivative (39), which was formed fromcommercially available Fmoc-aspartic acid α-allyl ester derivative (38)by protection of the β-ester under Mitsunobu conditions. The allyl esterwas removed using palladium (0) catalyst, followed by Weinreb amideformation using TBTU as the coupling agent. Diphenylmethyl3-Fmoc-amino-4-(methoxycarbonyl-substituted-methylamino)-butyrate (41)was then Fmoc deprotected, cyclized, diphenylmethyl ester removed byhydrogenation, followed by Fmoc protection to give the final product(4-Fmoc-5-substituted-6-oxo-piperazin-2-yl)-acetic acid (37).

Synthesis of Fmoc-Asp-(OCHPh₂) Weinreb amide (39): To a solution of 5.1g (13.0 mmol) of Fmoc-aspartic acid α-allyl ester (38) in 30 mL of drytetrahydrofuran, containing 3.4 g (13 mmol) of triphenylphosphine, and2.41 g (13.1 mmol) of diphenylmethanol, kept at 0° C. under nitrogen,was added slowly 2.6 mL (13.4 mmol) of diisopropyl azodicarboxylate. Theice bath was removed, and the reaction stirred at room temperatureovernight, concentrated to dryness, and then purified by silica gelcolumn chromatography. ¹H NMR δ (CDCl₃) 2.96-3.06 (dd, 1H, CH₂CO),3.15-3.26 (dd, 1H, CH₂CO), 4.18-4.76 (a series of m, 3H, CH, CH₂),5.14-5.32 (m, 1H, CHN), 5.76-5.86 (m, 1H, CHO), 7.20-7.80 (a series ofm, 18H, fulvene, and Ph); HPLC t_(R)=7.68 min, (M⁺+Na⁺)=583.90.

The product (9.8 mmol) was then dissolved in 40 mL of adichloromethane:acetic acid:N-methyl morpholine solution at 37:2:1,containing 1.5 g (1.3 mmol) of tetrakis triphenylphosphine palladium(0), and the solution stirred at room temperature overnight,concentrated to dryness, and partitioned between 100 mL of ethyl acetateand 30 mL of water. The layers were separated, and the organic layerwashed with 1×50 mL of water, dried over sodium sulfate, andconcentrated. The residue was suspended in 20 mL of dry dichloromethane,and 1.65 mL (15 mmol) of N-methyl morpholine, and 4.07 g (12.7 mmol) ofTBTU were added, and the suspension stirred at room temperature for 20minutes, followed by the addition of 1.65 mL (15 mmol) of N-methylmorpholine, and 1.52 g (15.6 mmol) of N,O-dimethyl hydroxylaminehydrochloride salt. The suspension was stirred at room temperature for 2hours, concentrated, partitioned between 100 mL of ethyl acetate and 50mL of water. The organic layer was washed with 1×30 mL of water, 1×30 mLof saturated sodium bicarbonate solution, and 1×30 mL of 1N hydrochloricacid solution, dried over sodium sulfate, and concentrated. The productwas purified by silica gel column chromatography. ¹H NMR δ (CDCl₃)2.76-2.88 (dd, 1H, CH₂CO), 2.89-3.00 (dd, 1H, CH₂CO), 3.16 (s, 3H,CH₃N), 3.70 (s, 3H, CH₃O), 4.14-4.22 (dd, 1H, CH), 4.28-4.40 (t, 2H,CH₂), 5.07-5.16 (dd, 1H, CHN), 5.69-5.76 (d, 1H, CHO), 7.24-7.8 (aseries of m, 18H, fulvene, and Ph); HPLC t_(R)=7.08, (M++Na⁺)=587.03.

Synthesis of Diphenylmethyl 3-Fmoc-amino-4-oxo-butyrate (40): Compound(40) is prepared using a procedure similar to the one described forcompound (9).

Synthesis of Diphenylmethyl3-Fmoc-amino-4-(methoxycarbonyl-substituted-methylamino)-butyrate (41):Compounds (41) were prepared using a procedure similar to the onedescribed for compound (10), but using compound (40) as the aldehyde.

R Analytical Data for Compounds (41)

¹H NMR δ (CDCl₃) 1.2-1.7 (m, 4 H, CH₂), 1.42 (s, 3 H, CH₃Ph), 1.60 (s, 6H, CH₃- Ph), 2.07 (s, 2 H, CH₂), 2.52 (s, 3 H, CH₃-Ph), 2.58 (s, 3 H,CH₃-Ph), 2.08-2.80 (a, series of m, 2 H, CH₂CO), 3.0-3.2 (m, 2 H, CH₂N),3.64 (s, 3 H, CH₃O), 3.96-4.10 (m, 1 H, CHN), 4.20-4.28 (m, 1 H, CH),4.28-4.40 (br. m, 2 H, CH₂), 5.82-6.18 (m, 1 H, CHO), 7.24-7.80 (aseries of m, 18 H, fulvene, and Ph), HPLC t_(R) = 6.53, (M⁺ + 1) =930.56.

Synthesis of (4-Fmoc-5-substituted-6-oxo-piperazin-2-yl)-acetic acid(37): A solution of 10 mmol of compound (41) in 30 mL of 30%diethylamine in ethyl acetate was stirred at room temperature for 3hours. The solution was then concentrated to dryness, redissolved in2×30 mL of ethyl acetate, and reconcentrated. The residue dissolved in50 mL of ethanol, and 20 mL of 1N hydrochloric acid solution, andhydrogenated at room temperature and atmospheric pressure overnight,filtered through celite, and concentrated to dryness. The residue wasdissolved in 20 mL of tetrahydrofuran, and 10 mL of water, and 2.52 g(30 mmol) of solid sodium bicarbonate was added, followed by theaddition of 3.3 g (13 mmol) of Fmoc-Cl. The mixture was stirred for 3hours, diluted with 100 mL of ethyl acetate, the layers separated, andthe organic layer washed with 2×50 mL of water, dried over magnesiumsulfate, and concentrated. The product was purified by silica gel columnchromatography.

R Analytical Data for Compounds (37)

¹H NMR δ (CDCl₃) 1.2-1.6 (m, and s, 7 H, CH₂, CH₃Ph), 2.10 (s, 2 H,CH₂), 2.46 (s, 3 H, CH₃-Ph), 2.56 (s, 3 H, CH₃-Ph), 2.46-2.63 (br. m, 2H, CH₂CO), 3.0-3.95 (3 b. m, 5 H, CH₂N, CHN), 4.10-4.30 (br. m, 1 H,CH), 4.40-4.80 (br. m, 3 H, CHN, CH₂), 7.22-7.80 (a series of m, 8 H,fulvene), HPLC t_(R) = 5.73, (M⁺ + 1) 732.24.

Method K: The syntheses of (5-substituted-6-oxo-piperazin-2-yl)-aceticacid scaffolds are done starting from commercially availableFmoc-Aspartic acid α tert-butyl ester (42). Fmoc-aspartic acid αtert-butyl ester is reduced to Fmoc-Homoserine α tert-butyl ester withsodium borohydride via the mixed anhydride, followed by protection ofthe alcohol with benzyl bromide to give Fmoc-Homoserine benzyl ether αtert-butyl ester (43). The tert-butyl ester is then removed withtrifluoroacetic acid, and the acid is reduced to the alcohol with sodiumborohydride via the mixed anhydride to give2-Fmoc-amino-4-benzyloxy-1-butanol (44). Alcohol (44) is then convertedto 2-Fmoc-amino-4-benzyloxybutanal (45) using Dess-Martin periodinane asdescribed previously. Reductive amination of2-Fmoc-amino-4-benzyloxybutanal (45) and α-amino ester (2) gives the(2-Fmoc-amino-4-benzyloxy-butylamino)-2-substituted acetic acid methylester (46). Fmoc deprotection with diethyl amine gives the free primaryamine which cyclizes to 6-benzyloxyethyl-3-substituted-piperazin-2-onespontaneously. The benzyl ether is removed by hydrogenation, and thesecondary amine is protected as its Fmoc derivative to give4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-ones (47). Finally, theprimary alcohol is oxidized to the acid to give the final products (48)as described in method A.

Synthesis of Fmoc-Homoserine (OBn) α tert-butyl ester (43): To asolution of 10.0 mmol of Fmoc Asp-O^(t)Bu (42) in 50 mL of drytetrahydrofuran, kept at −20° C. under nitrogen, is added 1.77 mL (12.7mmol) of triethyl amine, followed by the slow addition of 1.57 mL (12.0mmol) of isobutylchloroformate. The mixture is stirred for 30 minutes,and then poured slowly over an ice-cold solution of 3.77 g (99.6 m mol)of sodium borohydride in 10 mL of water, keeping the temperature below5° C. The reaction is stirred at 0° C. for 15 minutes, and then quenchedwith 1N hydrochloric acid solution. The reaction mixture is diluted with100 mL of ethyl acetate, and the layers separated. The organic layer waswashed with 2×25 mL of 1N hydrochloric acid solution, 2×25 mL of water,dried over magnesium sulfate and concentrated, and purified by silicagel column chromatography. Purified compound is then dissolved in 30 mLof tetrahydrofuran, and 12 mmol of 60% sodium hydride dispersion inmineral oil is added, followed by 0.2 mmol of tetrabutylammonium iodideand 12 mmol of benzyl bromide, and the mixture is stirred overnight,quenched with 50 mL of saturated aqueous sodium bicarbonate, andextracted with 100 mL of ethyl acetate. The compound is then purified bysilica gel column chromatography.

Synthesis of 2-Fmoc-amino-4-benzyloxy-1-butanol (44): Deprotection ofthe tert-butyl ester using 90% trifluoroacetic acid is done as describedfor compound (37) in method I, followed by reduction of the acid to thealcohol with sodium borohydride via the mixed anhydride intermediate asdescribed for compound (13).

Synthesis of 2-Fmoc-amino-4-benzyloxy-butanal (45):2-Fmoc-amino-4-benzyloxy-1-butanol (44) is oxidized to the aldehydeusing Dess-Martin periodinane as described for the synthesis of (9).

Synthesis of (2-Fmoc-amino-4-benzyloxy-butylamino)-2-substituted aceticacid methyl ester (46): reductive amination of2-Fmoc-amino-4-benzyloxy-butanal (45) with an α-amino ester (2) usingeither sodium cyanoborohydride or sodium triacetoxyborohydride as thereducing agent is done as described for the synthesis of (10).

Synthesis of 4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-ones (47):Fmoc deprotection of (2-Fmoc-amino-4-benzyloxy-butylamino)-2-substitutedacetic acid methyl ester (46) with concomitant cyclization, followed byde-benzylation and Fmoc reprotection is done as described for compound(37) in method J.

Synthesis of 4-Fmoc-5-substituted-6-oxo-piperazin-2-yl-acetic acid (37):Oxidation of 4-Fmoc-6-hydroxymethyl-3-substituted-piperazin-2-ones (47)to the acid is done as described in method A. The choice of theoxidizing agent used is based on the nature of the group in the5-position.

Synthesis of 2-Substituted 3-Oxo-[1,4]-diazepane-5-carboxylic acidScaffolds (Methods L, M, N)

The synthesis of 2-substituted 3-oxo-[1,4]-diazepane-5-carboxylic acidscaffolds is done using several methods.

Method L: tert-butyl2-Cbz-amino-4-(benzyloxycarbonyl-substituted-methyl-Boc amino)-butyrates(52) are prepared by reductive amination of tert-butylCbz-2-amino-4-oxo-butyrate (50) with amino ester (51), using eithersodium cyanoborohydride or sodium triacetoxyborohydride as the reducingagent, followed by Boc protection of the secondary amine. The tert-butylCbz-2-amino-4-oxo-butyrate (50) required for the reductive amination isprepared by lithium aluminum hydride reduction of the Weinreb amidederivative (49). The diazepane ring is formed by protecting groupremoval, followed by cyclization with a peptide forming reagent to give(53). Finally, 4-Fmoc-2-substituted 3-oxo-[1,4]-diazepane-5-carboxylicacids (54) are formed by protecting group exchange.

Synthesis of Cbz-Asp-(Weinreb amide)-O^(t)Bu (49): Compound (49) isprepared using a procedure similar to the one described for compound(14).

Synthesis of tert-butyl 3-Cbz-amino-4-oxo-butyrate (50): Compound (50)is prepared using a procedure similar to the one described for compound(9).

Synthesis of tert-butyl2-Cbz-amino-4-(benzyloxycarbonyl-substituted-methyamino)-butyrate (52):The reductive amination is done with procedure similar to the onedescribed for compound (10). The secondary amine is protected byreaction of the crude mixture with 2 equivalents of Boc dicarbonate intetrahydrofuran.

Synthesis of tert-butyl 1-Boc2-substituted-3-oxo-[1,4]-diazepane-5-carboxylate (53): A solution of 10mmol of compound (52) in 30 mL of ethanol is hydrogenated at roomtemperature and atmospheric pressure for 2 hours, filter through celite,and concentrated to dryness. The residue is dissolved in 100 mL ofdichloromethane and 1.2 equivalents of TBTU, and 2.6 equivalents ofN-methyl-morpholine are added. The solution is stirred at roomtemperature overnight, and then concentrated. The residue is partitionedbetween 50 mL of ethyl acetate and 25 mL of 1N hydrochloric acidsolution, washed with 1×20 mL of a saturated sodium bicarbonatesolution, dried over magnesium sulfate, and concentrated.

Synthesis of 1-Fmoc 2-substituted-3-oxo-[1,4]-diazepane-5-carboxylicacid (54): A solution of 10 mmol of compound (53) in 10 mL of 90%trifluoroacetic acid in dichloromethane is stirred at room temperaturefor 2 hours, and then the solution is concentrated to dryness. Theresidue is dissolved in 20 mL of tetrahydrofuran and 10 mL of water, and2.52 g (30 mmol) of solid sodium bicarbonate is added, followed by theaddition of 3.36 g (13 mmol) of Fmoc-Cl. The mixture is stirred for 3hours, and then diluted with ethyl acetate. The layers are separated,and the organic layer washed with 2×50 mL of water, dried over magnesiumsulfate, and concentrated.

Method M: the reduced dipeptide analogs (60) are prepared by reductiveamination of diphenylmethyl Alloc-2-amino-4-oxo-butyrate (59) with aminoester (29), using either sodium cyanoborohydride or sodiumtriacetoxyborohydride as the reducing agent, followed by Cbz protectionof the secondary amine. Diphenylmethyl Alloc-2-amino-4-oxo-butyrate (59)required for the reductive amination is prepared by lithium aluminumhydride reduction of the Weinreb amide derivative (58), which isprepared by protecting group exchange of Weinreb amide derivative (57).The diazepane ring is then formed by allyl and alloc group removal,followed by ring closure in the presence of a peptide forming reagent.2-substituted 3-oxo-[1,4]-diazepane-5-carboxylic acid scaffolds (54) areformed by protecting group exchange.

Synthesis of Fmoc-Asp-(Weinreb amide)-OCHPh₂ (57): Compound (57) isprepared using a procedure similar to the one described for compound(39).

Synthesis of Alloc-Asp-(Weinreb amide)-OCHPh₂ (58): A solution of 10mmol of compound (56) in 20 mL of 30% diethylamine in ethyl acetate isstirred for 2 hours, and concentrated to dryness. The residue isdissolved in 20 mL of tetrahydrofuran and 10 mL of water, and 2.52 g (30mmol) of solid sodium bicarbonate is added, followed by the addition of13 mmol of Alloc-Cl. The mixture is stirred for 3 hours, and thendiluted with ethyl acetate. The layers are separated, and the organiclayer washed with water, dried over magnesium sulfate, and concentrated.Compound (58) is purified by silica gel column chromatography.

Synthesis of diphenylmethyl 3-Alloc-amino-4-oxo-butyrate (59): Compound(59) is prepared using a procedure similar to the one described forcompound (9).

Synthesis of diphenyl methyl2-Alloc-amino-4-(allyloxycarbonyl-substituted-methyamino)-butyrate (60):compound 60 is prepared by reductive amination using a procedure similarto the one described for compounds (15), but using compound (59) as thealdehyde. The product is purified by silica gel column chromatography.

Synthesis of diphenylmethyl 1-Cbz2-substituted-3-oxo-[1,4]-diazepane-5-carboxylate (61): To a solution of10 mmol of compound (60) in 30 mL of dichloromethane, kept at roomtemperature under nitrogen, is added 2 equivalents of phenylsilane and0.3 equivalents of tetrakistriphenylphosphine palladium (0), and thesolution stirred for 2 hours, and then 1.2 equivalents of TBTU and 1.3equivalents of N-methyl-morpholine are added. The solution is stirred atroom temperature overnight and concentrated. The residue is partitionedbetween 50 mL of ethyl acetate and 25 mL of 1N hydrochloric acidsolution, washed with 1×20 mL of a saturated sodium bicarbonatesolution, dried over magnesium sulfate, and concentrated.

Synthesis of 1-Fmoc 2-substituted-3-oxo-[1,4]-diazepane-5-carboxylicacid (54): A solution of 10 mmol of compound (61) in 30 mL of ethanol ishydrogenated at room temperature for 2 hours, filtered through celite,and then the solution is concentrated to dryness. The residue isdissolved in 20 mL of tetrahydrofuran, and 10 mL of water, and 2.52 g(30 mmol) of solid sodium bicarbonate is added, followed by the additionof 3.36 g (13 mmol) of Fmoc-Cl. The mixture is stirred for 3 hours, andthen diluted with ethyl acetate. The layers are separated, and theorganic layer washed with water, dried over magnesium sulfate, andconcentrated.

Method N: Fmoc-Aspartic acid β tert-butyl ester is reduced toFmoc-Aspartanol β tert-butyl ester (63) with sodium borohydride via themixed anhydride, followed by protection of the alcohol with allylbromide to give Fmoc-Aspartanol allyl ether β tert-butyl ester (64). Thetert-butyl ester is then removed with trifluoroacetic acid, and the acidreduced to the alcohol with sodium borohydride via the mixed anhydrideto give 3-Fmoc-amino-4-allyloxy-1-butanol (65). Alcohol (65) is thenconverted to 3-Fmoc-amino-4-allyloxybutanal (66) using Dess-Martinperiodinane as described previously. Reductive amination of3-Fmoc-amino-4-allyloxybutanal (66) and α amino ester (51), followed byalloc protection on the secondary amine, gives the(3-Fmoc-amino-4-allyloxy-butyl-alloc-amino)-2-substituted acetic acidbenzyl esters (67). Alloc7-allyloxymethyl-3-substituted-[1,4]-diazepan-2-ones (68) are formed bysaponification of the benzyl ester, followed by Fmoc deprotection withdiethyl amine to give the free primary amine which is cyclized using apeptide forming reagent such as TBTU. The final products (54) are formedby protecting group exchange: the allyl ether and the alloc are removedby palladium (0), and the secondary amine is protected as its Fmocderivative to give4-Fmoc-7-benzyloxymethyl-3-substituted-[1,4]-diazepan-2-ones, followedby primary alcohol oxidation to the acid to give the final products(54). The choice of the oxidizing agent used is based on the nature ofthe group in the 2-position.

Synthesis of Fmoc-Aspartanol β tert-butyl ester (63): Compound (63) isprepared as described for the synthesis of compound (13), usingFmoc-Aspartic acid β tert-butyl ester (62) as the starting material.

Synthesis of 3-Fmoc-amino-4-allyloxy-butyric acid tert-butyl ester (64):To a solution of 10 mmol of (63) in 30 mL of tetrahydrofuran, kept atroom temperature under nitrogen, is added 12 mmol of 60% sodium hydridedispersion in mineral oil, 2 mmol of tetrabutylammonium iodide, and 13mmol allyl bromide, and the mixture is stirred overnight, quenched with10 mL of saturated aqueous sodium bicarbonate, and extracted with 50 mLof ethyl acetate.

Synthesis of 3-Fmoc-amino-4-allyloxy-1-butanol (65): Compound (65) isprepared as described for the synthesis of compound (44).

Synthesis of 3-Fmoc-amino-4-allyloxy-butanal (66):3-Fmoc-amino-4-allyloxy-1-butanol (65) is oxidized to the aldehyde usingDess-Martin periodinane as described for the synthesis of (9).

Synthesis of (3-Fmoc-amino-4-allyloxy-butyl-alloc-amino)-2-substitutedacetic acid methyl ester (67): reductive amination of3-Fmoc-amino-4-benzyloxy-butanal (66) with an α-amino ester (51) usingeither sodium cyanoborohydride or sodium triacetoxyborohydride as thereducing agent as described for compound (10), followed by protection ofthe secondary amine as the alloc derivative, is done as described forcompound (15), but using allyl chloroformate instead of benzylchloroformate.

Synthesis of4-Alloc-7-allyloxymethyl-3-substituted-[1,4]-diazepan-2-ones (68): Asolution of 10 mmol of(3-Fmoc-amino-4-allyloxy-butyl-alloc-amino)-2-substituted acetic acidmethyl ester (67), 20 mmol of potassium carbonate in 20 mL of methanol,and 10 mL of water is stirred at room temperature for 3 hours,neutralized with 21 mL of a 1N hydrochloric acid solution, and thenconcentrated to dryness. The residue is dissolved in 20 mL of 30%diethyl amine in ethyl acetate and stirred at 3 hours, and thenconcentrated to dryness. The residue is dissolved in 100 mL ofdichloromethane, and 12 mmol of TBTU and 24 mmol of N-methylmorpholineare added, and the solution stirred at room temperature overnight, andthen concentrated to dryness. The residue is partitioned between 30 mLof ethyl acetate and 30 mL of 1N hydrochloric acid solution, and thenthe layers separated. The organic layer is washed with 30 mL of asaturated sodium bicarbonate solution, dried over magnesium sulfate, andpurified by silica gel column chromatography.

Synthesis of 4-Fmoc-2-substituted-3-oxo-[1,4]-diazepane-5-carboxylicacid (54): To solution of 10 mmol of compound (68) in 30 mL ofdichloromethane, kept at room temperature under nitrogen, is added 2equivalents of phenylsilane and 0.3 equivalents oftetrakistriphenylphosphine palladium (0), and the solution then stirredfor 2 hours, and concentrated to dryness. The secondary amine isdissolved in 20 mL of tetrahydrofuran, and 10 mL of water, followed bythe addition of 2.52 g (30 mmol) of solid sodium bicarbonate, and 1.2equivalents of Fmoc-Cl and the biphasic solution is stirred at roomtemperature for 2 hours, diluted with 30 mL of ethyl acetate, and thelayers separated. Oxidation of4-Fmoc-7-hydroxymethyl-3-substituted-[1,4]-diazepan-2-ones to the finalproduct (54) is done as described in method A. The choice of theoxidizing agent used is based on the nature of the group in the2-position, as in Method A for the conversion of (6) to (7).

Synthesis of 6-substituted-5-oxo-piperazine-2-carboxylic acid Scaffolds(Method O)

The syntheses of 6-substituted-5-oxo-piperazine-2-carboxylic acidscaffolds containing non-functionalized side chains in the 6-positionare done as outlined in Method O, starting from commercially available3-Fmoc-amino-1,2-propan-diol 1-chloro-trityl resin (69) which isoxidized to the ketone (70) using Dess-Martin periodinane. Reductiveamination of ketone (70) with an α amino ester (2) gives resin bound(1-aminomethyl-2-chloro-trityloxy-ethylamino)-2-substituted acetic acidmethyl ester (71), which is cyclized to5-chlorotrityloxymethyl-3-substituted-piperazin-2-one (72) afterdeprotection of the amine. Reprotection of the secondary amine, followedby cleavage from the resin, givesFmoc-5-hydroxymethyl-3-substituted-piperazin-2-one (73) which isoxidized to 6-substituted-5-oxo-piperazine-2-carboxylic acid (74) usingeither of the procedures described in method A.

Synthesis of 1-amino-3-chlortrityloxy-propan-2-one (70): the oxidationof resin bound alcohol (69) is done by sulfur trioxide oxidation,NMO/TPAP (N-methylmorpholine-N-oxide/tetrapropyl ammonium perrthenate)oxidation, or PDC oxidation. For sulfur trioxide oxidation, a proceduresimilar to the one described in Parikh, J. R. and Doering, W. V., J. Am.Chem. Soc. 89:5505-5507 (1967) is used. For NMO/TPAP oxidation, to 0.3mmol of resin-bound alcohol is added a solution of 3 mmol ofN-methylmorpholine N-oxide in 10 mL of dry dimethylformamide, and then0.06 mmol of tetrapropylammonium perruthenate (TPAP) is added to theresin suspension. The reaction is shaken for 80 minutes. The solvent isdrained, the resin washed with tetrahydrofuran and dichloromethane, andthen dried under vacuum. For PDC oxidation, a suspension of resin boundalcohol in 0.2 M pyridinium dichromate in dimethylformamide is shaken at37° C. for 4 hours, the solvent is drained, and the resin washed withdimethylformamide, tetrahydrofuran, and dichloromethane.

Synthesis of (1-aminomethyl-2-chloro-trityloxy-ethylamino)-2-substitutedacetic acid methyl ester (71): the reductive amination of resin boundketone (70) with amino ester is done by one of two different methods. Inone method, a solution of 2.6 mmol of α amino ester (2) in 20 mL of 1%acetic acid in dimethylformamide is added 2.6 mmol of sodiumtriacetoxyborohydride, followed by the immediate addition of 0.5 mmol ofketone-derivatized resin (70), and the mixture is shaken for 60 minutes,rinsed with methanol, 10% di-isopropyl ethyl amine, dimethylformamide,and methanol. In a second method, a suspension of 0.05 mmol ofketone-derivatized resin (70) and 2.0 M α amino ester hydrochloride (2)in methanol, containing 0.05 M sodium cyanoborohydride is shaken at roomtemperature for 5 hours, drained, and washed.

Synthesis of 5-chlorotrityloxymethyl-3-substituted-piperazin-2-one (72):A suspension of 0.05 mmol of resin in 10 mL of 20% piperidine indimethylformamide is shaken at room temperature for 2 hours.

Synthesis of Fmoc-5-hydroxymethyl-3-substituted-piperazin-2-one (73): Asuspension of 0.05 mmol of (72) in 10 mL of dichloromethane, containing0.25 mmol of Fmoc-Cl and 0.25 mmol of triethyl amine is stirred at roomtemperature for 6 hours, drained, and washed with dichloromethane. Theresin is resuspended in 10 mL of 95% trifluoroacetic acid indichloromethane, and the suspension shaken for 2 hours, and filtered,and the filtrate is concentrated.

Synthesis of Fmoc-6-substituted-5-oxo-piperazine-2-carboxylic acid (74):Oxidation of (73) to the desired product is done by any of theprocedures described for method A.

Synthesis of α, α-Disubstituted Amino Acids (Methods P and Q)

In certain of the constructs of the invention, it is possible andcontemplated to employ a disubstituted amino acid residue, such as an α,α-disubstituted amino acid where the substituents are either the same ordifferent. In one aspect, an α, α-disubstituted amino acid is employedin either the Aaa¹ or Aaa⁸ position, wherein at least one of the sidechains of the α, α-disubstituted amino acid is a side chain of Nle, Ala,Leu, Ile, Val, Nva, Met(O) or Met(O₂). The following synthetic Methods Pand Q describe making α, α-di-n-butylglycine (2-Amino-2-butyl-hexanoicacid), wherein each of the side chains are —(CH₂)₃—CH₃, and thus each isthe same as the side chain of Nle. However, it is to be understood thatsimilar methods and schemes may be employed in the making of other α,α-disubstituted amino acids, where the substituents are either the sameor different. Additionally, any method of making an α, α-disubstitutedamino acid may be employed in the practice of this invention, and thepractice of this invention is not limited to the methods of thefollowing synthetic schemes. Thus any method known in the art for thesynthesis of α, α-disubstituted amino acids may be employed in thepractice of this invention. The following teach alternative methods forthe making of α, α-disubstituted amino acids: Clark J. S. and MiddletonM. D.: Synthesis of novel alpha-substituted andalpha,alpha-disubstituted amino acids by rearrangement of ammoniumylides generated from metal carbenoids. Org. Lett. 4(5):765-8 (2002);Guino M., Hii K. K.: Wang-aldehyde resin as a recyclable support for thesynthesis of alpha,alpha-disubstituted amino acid derivatives. Org.Biomol. Chem. 3(17):3188-93 (2005); and Kotha S., Behera M.: Synthesisand modification of dibenzylglycine derivatives via the Suzuki-Miyauracross-coupling reaction. J. Pept. Res. 64(2):72-85 (2004).

Synthesis of Benzoyl di-n-butylglycine (80): To a solution of 10 mmolbenzoyl glycine (75) in 20 mL of dichloromethane, kept at 0° C. undernitrogen, is added slowly 12 mmol of N,N′-dicyclohexylcarbodiimide(DCC), and the reaction stirred for 2 hours to yield compound (76). Thesolid is filtered off, and the filtrate concentrated. The residue isdissolved in 15 mL of tetrahydrofuran, cooled to 0° C., and then 24 mmolof sodium hydride is added, followed by 30 mmol of n-butyl bromide. Thesuspension is stirred at 0° C. for 2 hours and then allowed to warm toroom temperature, and the solution concentrated to dryness to yieldcompound (77). Alternatively, compound (77) can also be prepared frombenzoyl norleucine (78) in a similar manner except that 12 mmol ofsodium hydride and 15 mmol of n-butyl bromide are used. Compound (77) isdissolved in methanol, 50 mL of 1N hydrochloric acid solution is added,and the solution stirred for 2 hours, and concentrated. Compound (80) ispurified by silica gel column chromatography.

Synthesis of Fmoc di-n-butylglycine (81): 10 mmol of compound (80) isdissolved in 30 mL of dioxane, and 10 mL of 6N hydrochloric acidsolution is added, and the solution is refluxed overnight. The reactionis cooled to room temperature, concentrated to dryness, redissolved in30 mL of tetrahydrofuran, and 10 mL of water and 30 mmol of sodiumbicarbonate is added, followed by 15 mmol of Fmoc-Cl. The biphasicsolution is stirred for 1 hour, and the tetrahydrofuran removed undervacuum. The aqueous solution is extracted with 1×50 mL of diethyl ether,acidified with 1N hydrochloric acid solution, and extracted with 2×50 mLof ethyl acetate. The ethyl acetate layers are combined, dry over sodiumsulfate, and concentrated. Compound (81) is purified by silica gelcolumn chromatography.

Similar methods may be employed by starting with any appropriate aminoacid derivative (similar to compound 78), and by using an appropriatealkyl butyl, aryl butyl, or aralkyl butyl reagent the scheme will yielda variety of disubstituted (R, R′) amino acid surrogates where R and R′are different.

Synthesis of Fmoc—α,α di-n-butyl glycine (87): To a suspension of 20mmol of glycine methyl ester hydrochloride (82), and 2 g of powderedmolecular sieves in 40 mL of dry tetrahydrofuran, kept at roomtemperature, is added 24 mmol of potassium hydroxide, followed by 22mmol of benzaldehyde. The suspension is stirred for 2 hours, filtered,and the filtrate concentrated. The residue is redissolved in 40 mL ofdry toluene, and then added to a suspension of 60 mmol of sodium hydridein toluene, followed by the addition of 60 mmol of n-butyl bromide. Thesuspension is stirred for 12 hours, followed by addition of 30 mL of asolution of 6N hydrochloric acid, stirred at room temperature for 2hours, and then the layers separated. The hydrochloride salt of (84)thus obtained is used in situ for preparation of (87). To isolate (84)as the hydrochloride salt the aqueous layer is concentrated to drynessand the product crystallized from dry methanol-ether.

Alternatively, compound (84) can be prepared from norleucine methylester hydrochloride using a similar synthetic procedure except that 30mmol of sodium hydride and 30 mmol of n-butyl bromide are used forconversion of (86) to (84).

The aqueous mixture of the hydrochloride form of compound (84) asobtained above is heated to reflux for 1 hour and then cooled to roomtemperature. It is neutralized with solid sodium hydroxide and thendiluted with 30 mL of tetrahydrofuran. Sodium bicarbonate (30 mmol) isadded followed by 15 mmol of Fmoc-Cl. The biphasic solution is stirredfor 1 hour, and the tetrahydrofuran removed under vacuum. The aqueoussolution is extracted with 1×50 mL of diethyl ether, acidified with 1Nhydrochloric acid solution, and extracted with 2×50 mL of ethyl acetate.The ethyl acetate layers are combined, dried over sodium sulfate, andconcentrated. Compound (87) is purified by silica gel columnchromatography.

Similar methods may be employed by starting with any appropriate aminoacid derivative (similar to compound 85), and by using an appropriatealkyl butyl, aryl butyl, or aralkyl butyl reagent the scheme will yielda variety of disubstituted (R, R′) amino acid surrogates where R and R′are different.

Synthesis of Disubstituted (R, R′) Scaffolds (Method R)

The invention further provides for constructs in which amino acidsurrogates are employed with two R groups, R and R′. The followingmethod describes synthesis of Fmoc protected(R)-5,5-dibutyl-6-oxo-piperazine-2-carboxylic acid, where R and R′ areeach groups corresponding to a norleucine side chain moiety. It may beseen that the method below may be modified, based in part on theforegoing methods, to produce similar disubstituted (R, R′) amino acidsurrogates. Similar methods may be employed such that starting with anyappropriate amino acid derivative (a compound similar to compound (84))the scheme can yield a variety of disubstituted (R, R′) amino acidsurrogates where R and R′ are different.

Synthesis of (2-Fmoc-amino-3-tert-butoxy-propylamino)-2,2,di-n-butylacetic acid methyl ester (88): A suspension of 21 mmol of (84, schemeQ), and 2.9 mL (21 mmol) of triethyl amine in 50 mL of drytetrahydrofuran, is stirred at room temperature for 45 minutes, and thena solution of ˜20 mmol crude Fmoc-(O-t-butyl)-serinal (9, scheme D) in30 mL of tetrahydrofuran is added, followed by 1.7 g of 4 Å powderedmolecular sieves, and the suspension is stirred for an additional 2hours. 6.4 g (30 mmol) of solid sodium triacetoxyborohydride is added,and the suspension stirred at room temperature overnight. The suspensionis diluted with methanol, the molecular sieves filtered, and thefiltrate concentrated. The residue is partitioned between 100 mL ofethyl acetate and 50 mL of water. The organic layer is dried over sodiumsulfate, filtered, and concentrated. Compound (88) is purified by silicagel column chromatography.

Synthesis of 4-Fmoc-6-hydroxymethyl-3,3-di-n-butyl-piperazin-2-one (89):A solution of 10 mmol of compound (88) in 30 mL of 30% diethyl amine inethyl acetate is stirred at room temperature overnight, and thenconcentrated to dryness. The residue is dissolved in 20 mL oftetrahydrofuran and 10 mL of water, 2.52 g (30 mmol) of solid sodiumbicarbonate is added, followed by 3.36 g (13 mmol) of Fmoc-Cl. Themixture is stirred for 3 hours, diluted with 50 mL of ethyl acetate, thelayers separated, and the organic layer washed with 30 mL of water,dried over magnesium sulfate, and concentrated. The crude mixture isdissolved in a solution of 10 mL of 90% trifluoroacetic acid indichloromethane, stirred for 2 hours, and then concentrated to dryness.The residue is dissolved in ethyl acetate and washed with 50 mL of asaturated solution of sodium bicarbonate, dried over magnesium sulfate,and concentrated. Compound (89) is purified by silica gel columnchromatography.

Synthesis of 4-Fmoc-5,5-di-n-butyl-6-oxo-piperazine-2-carboxylic acid(90): To a solution of 8 mmol alcohol (89) in 81 mL of acetonitrile keptat room temperature, is added phosphate buffer solution (prepared with0.72 g of sodium phosphate monobasic and 1.43 g of sodium phosphatedibasic in 29.5 mL of water), followed by the addition of 0.33 g (2.1mmol) of TEMPO, and 1.86 g (16.5 mmol) of sodium chlorite, and thebiphasic solution is placed in an oil bath kept at 43° C. A solution of4.3 mL (2.6 mmol) of sodium hypochlorite solution (prepared by mixing1.9 mL of 10-13% sodium hypochlorite solution, and 2.4 mL of water) isadded slowly. The reaction is stirred at 43° C. for 4 hours, cooled toroom temperature, 20 mL of 10% sodium hydrogen sulfite added, stirredfor 10 minutes, diluted with 50 mL of ethyl acetate, and the layersseparated. The organic layer is washed with 1×10 mL of brine, 1×10 mL of1N hydrochloric acid solution, dried over sodium sulfate, andconcentrated. Compound (90) is purified by silica gel columnchromatography.

Synthesis of Constructs of the Invention

The constructs as disclosed in the several embodiments of this inventionmay be readily synthesized by any known conventional procedure for theformation of a peptide linkage between amino acids. Such conventionalprocedures include, for example, any solution phase procedure permittinga condensation between the free alpha amino group of an amino acidresidue having its carboxyl group or other reactive groups protected andthe free primary carboxyl group of another amino acid residue having itsamino group or other reactive groups protected. In a preferredconventional procedure, the constructs of this invention may besynthesized by solid-phase synthesis and purified according to methodsknown in the art. The amino acid surrogates of the present invention maybe incorporated into constructs of this invention by methodssubstantially similar to or identical to those employed with residues.Any of a number of well-known procedures utilizing a variety of resinsand reagents may be used to prepare the constructs of this invention.

The process for synthesizing the constructs may be carried out by aprocedure whereby each amino acid or amino acid surrogate in the desiredsequence is added one at a time in succession to another amino acidresidue or amino acid surrogate or by a procedure whereby peptidefragments with the desired amino acid sequence, which may include one ormore amino acid surrogates, are first synthesized conventionally andthen condensed to provide the desired construct. The resulting constructis cyclized to yield a cyclic construct of the invention.

Solid phase peptide synthesis methods are well known and practiced inthe art. In such methods the synthesis of constructs of the inventioncan be carried out by sequentially incorporating the desired amino acidresidues or amino acid surrogates one at a time into the growing peptidechain according to the general principles of solid phase methods. Thesemethods are disclosed in numerous references, including Merrifield R.B., Solid phase synthesis (Nobel lecture). Angew. Chem. 24:799-810(1985) and Barany et al., The Peptides, Analysis, Synthesis and Biology,Vol. 2, Gross E. and Meienhofer J., Eds. Academic Press, 1-284 (1980).

In chemical syntheses of constructs, reactive side chain groups of thevarious amino acid residues or amino acid surrogates are protected withsuitable protecting groups, which prevent a chemical reaction fromoccurring at that site until the protecting group is removed. Alsocommon is the protection of the alpha amino group of an amino acidresidue or amino acid surrogate while that entity reacts at the carboxylgroup, followed by the selective removal of the alpha amino protectinggroup to allow a subsequent reaction to take place at that site.Specific protecting groups have been disclosed and are known in solidphase synthesis methods and solution phase synthesis methods.

Alpha amino groups may be protected by a suitable protecting group,including a urethane-type protecting group, such as benzyloxycarbonyl(Z) and substituted benzyloxycarbonyl, such asp-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl,9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz);aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl(Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl, andallyloxycarbonyl. Fmoc is preferred for alpha amino protection.

Guanidino groups may be protected by a suitable protecting group, suchas nitro, p-toluenesulfonyl (Tos), Z, pentamethylchromanesulfonyl (Pmc),adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf),Fmoc and Boc. Pbf is one preferred protecting group for Arg. Otherpreferred protecting groups include Z, Fmoc, and Boc. It is to beunderstood that particularly guanidino protecting groups may be cleavedand removed during the synthetic process, or may alternatively not becleaved or removed, in which event the side chain with the protectinggroup forms a derivative of an amino acid side chain moiety as definedherein. Particularly where the protecting group is labile, and may beremoved by some mechanism in vivo upon administration to a patient, theconstruct becomes a “prodrug”, which is to say a construct that is adrug precursor which, following administration to a patient, isconverted to the desired drug form in vivo via some chemical orphysiological process (e.g., a prodrug on being brought to physiologicalpH or through enzyme action is converted to the desired drug form).

The constructs of the invention described herein can be prepared usingsolid phase synthesis, either manually or by means of an automatedpeptide synthesizer, using programming modules as provided by themanufacturer and following the protocols set forth by the manufacturer,or by modifications of the manufacturers's protocols to improve theyield of difficult couplings.

Solid phase synthesis is commenced from the C-terminal end of theconstruct by coupling a protected α-amino acid, α-amino acid surrogateor α-amino alcohol mimetic to a suitable resin. Such starting materialis prepared by attaching an α-amino-protected amino acid orα-amino-protected amino acid surrogate by an ester linkage to ap-benzyloxybenzyl alcohol (Wang) resin or a 2-chlorotrityl chlorideresin, by an amide bond between an Fmoc-Linker, such as p-[(R,S)-α-[1-(9H-fluor-en-9-yl)-methoxyformamido]-2,4-dimethyloxybenzyl]-phenoxyaceticacid (Rink linker) to a benzhydrylamine (BHA) resin, or by other meanswell known in the art, such as by attaching an α-amino-protected alcoholmimetic to 3,4-dihydro-2H-pyran-2yl-methanol linker attached tochloromethyl polystyrene resin. Fmoc-Linker-BHA resin supports arecommercially available and generally used when feasible. The resins arecarried through repetitive cycles as necessary to add amino acidssequentially. The alpha amino Fmoc protecting groups are removed underbasic conditions. Piperidine, piperazine, diethylamine, or morpholine(20-40% v/v) in N,N-dimethylformamide (DMF) may be used for thispurpose.

Following removal of the alpha amino protecting group, the subsequentprotected amino acids or amino acid surrogates are coupled stepwise inthe desired order to obtain an intermediate, protected peptide-resin.The activating reagents used for coupling of the amino acids in thesolid phase synthesis of the peptides are well known in the art. Afterthe construct is synthesized, if desired, the orthogonally protectedside chain protecting groups may be removed using methods well known inthe art for further derivatization of the construct.

Reactive groups in a construct can be selectively modified, eitherduring solid phase synthesis or after removal from the resin. Forexample, constructs can be modified to obtain N-terminus modifications,such as acetylation, while on resin, or may be removed from the resin byuse of a cleaving reagent and then modified. Methods for N-terminusmodification, such as acetylation, or C-terminus modification, such asamidation or introduction of an N-acetyl group, are known in the art.Similarly, methods for modifying side chains of amino acids are wellknown to those skilled in the art of peptide synthesis. The choice ofmodifications made to reactive groups present on the construct will bedetermined, in part, by the characteristics that are desired in theconstruct.

The construct are, in one embodiment, cyclized prior to cleavage fromthe resin. For cyclization through reactive side chain moieties, thedesired side chains are deprotected, and the construct suspended in asuitable solvent and a cyclic coupling agent added. Suitable solventsinclude, for example DMF, dichloromethane (DCM) or1-methyl-2-pyrrolidone (NMP). Suitable cyclic coupling reagents include,for example, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU),benzotriazole-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate(BOP),benzotriazole-1-yl-oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate(PyBOP), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TATU),2-(2-oxo-1(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TPTU) or N N′-dicyclohexylcarbodiimide/1-hydroxybenzotriazole(DCCl/HOBt). Coupling is conventionally initiated by use of a suitablebase, such as N,N-diispropylethylamine (DIPEA), sym-collidine orN-methylmorpholine (NMM).

Following cleavage of constructs from the solid phase followingsynthesis, the construct can be purified by any number of methods, suchas reverse phase high performance liquid chromatography (RP-HPLC), usinga suitable column, such as a C₁₈ column. Other methods of separation orpurification, such as methods based on the size or charge of theconstruct, can also be employed. Once purified, the construct can becharacterized by any number of methods, such as high performance liquidchromatograph (HPLC), amino acid analysis, mass spectrometry, and thelike.

Constructs of the present invention with a substituted amide derivativeC-terminus, typically an N-alkyl group, are prepared by solid phasesynthesis commenced from the C-terminal end of the construct by couplinga protected alpha amino acid or amino acid surrogate to a suitableresin. Such methods for preparing substituted amide derivatives on solidphase have been described in the art. See, for example, Barn D. R.,Morphy J. R., Rees D. C. Synthesis of an array of amides by aluminumchloride assisted cleavage of resin-bound esters. Tetrahedron Lett. 37,3213-3216 (1996); DeGrado W. F. Kaiser E. T. Solid-phase synthesis ofprotected peptides on a polymer bound oxime: Preparation of segmentscomprising the sequences of a cytotoxic 26-peptide analogue. J. Org.Chem. 47:3258-3261 (1982). Such starting material can be prepared byattaching an alpha amino-protected amino acid or amino acid surrogate byan ester linkage to a p-benzyloxybenzyl alcohol (Wang) resin by wellknown means. The peptide chain is grown with the desired sequence ofamino acids or amino acid surrogates, the product cyclized andresin-treated with a solution of appropriate amine and aluminum chloride(such as methyl amine, dimethyl amine, ethylamine, and so on) indichloromethane. The resulting amide derivative construct is released insolution from the resin. The resin is filtered and the amide derivativeconstruct recovered by concentration of solvent followed byprecipitation with ether. The crude construct is dried and remainingamino acid side chain protective groups cleaved using trifluoroaceticacid (TFA) in the presence of water and triisopropylsilane (TIS). Thefinal product is precipitated by adding cold ether and collected byfiltration. Final purification is by RP-HPLC using a C₁₈ column.

In one preferred method, the constructs of Example 1 were synthesized bythe following methods. Each of the constructs had one or two amino acidsurrogates based on a keto-piperazine structure. The amino acidsurrogates were synthesized as described above. The constructs weresynthesized using Fmoc chemistry. A manual synthetic approach was usedfor couplings immediately before and after incorporation of theketo-piperazine amino acid surrogate.

The following protocol was employed to attach an amino acid surrogate toresin, such as where the amino acid surrogate was in a terminalposition. Rink amide resin (loading at 0.3 mmol/g, Advanced ChemTech)was allowed to swell in DMF for 30 minutes. Fmoc deprotection of theresin was accomplished using 20% piperidine/DMF for 20 minutes. Couplingof the resin with the selected Fmoc-protected keto-piperazine amino acidsurrogate (2 eq) was accomplished by overnight incubation in DMF withPyBop (2 eq) and DIEA (4 eq). If following Kaiser testing a positiveresult was obtained, the coupling reaction was conducting a second time.Acetylation was carried out using Ac₂O (10 eq) and pyridine (20 eq) inDMF.

The following protocol was employed to attach a keto-piperazine aminoacid surrogate to peptide-resin. Coupling was carried out by mixingFmoc-protected keto piperzine amino acid surrogate (2 eq), TBTU (2 eq)and DIEA (4 eq) in DMF and allowing to incubate overnight, again with arepeat of the coupling reaction if a positive Kaiser test obtained.Acetylation was carried out using Ac₂O (10 eq) and pyridine (20 eq) inDMF.

The following protocol was employed to couple an Fmoc-protected aminoacid to a keto-piperazine amino acid surrogate on solid phase. In mostinstances at least two coupling cycles were needed, and frequently threecycles were employed. In a typical cycle Fmoc-protected amino acid (4eq) was mixed with HOAt (4 eq) and DIC (4 eq) in DMF. The resultingmixture was then mixed overnight in a SPE tube with a keto-piperazineamino acid surrogate attached directly or through intermediates toresin.

Couplings between amino acids that were not directly adjacent to aketo-piperazine amino acid surrogate in the sequence were conductedusing standard protocols for solid phase peptide synthesis. Thefollowing protecting groups were employed: Boc for Lys and Orn, t-Butylfor Tyr and Ser, Trityl for Cys and His, O-t-Butyl for Asp and Pbf forArg.

Constructs were cleaved from resin employing a mixture ofTFA/thioanisole/phenol/H₂O/DTT/TIS (87.5/2.5/2.5/5/2.5/11) (5 mL) for 3hours. The resulting material was filtered and precipitated from coldether under freezing conditions for one hour. Precipitated cysteinylpeptide was washed with cold ether at least three times before being usein an oxidation step.

For cyclization to form disulfide bonds via air oxidation, crudecysteinyl construct was dissolved in a mixture of acetonitrile andwater. The pH of the reaction mixture was adjusted to 7-8 using 5%NH₄OH. The resulted solution was stirred slowly with 150 mg granularactivated carbon for 2 days. Completion of cyclization was confirmed byLC-MS analysis before proceding to the next process step. Aftercyclization, solid carbon was filtered from solution. The filtrate waslyophilized or dried in a speed-vac to obtain crude cyclic construct.

Certain constructs of the invention, where the surrogate is bound toresin or other peptide solid support and is at the C-terminal position,may be synthesized by means of the following scheme. The followingscheme is exemplified by synthesis of construct 1-132, but it is to beunderstood that substantially similar methods may be employed for anyconstruct wherein the surrogate is bound to resin or other peptide solidsupport.

Surrogate (7) is prepared by the scheme of method A above, or anyalternative method. Fmoc protected Sieber amide resin was treated byswelling 23.8 g (0.63 mmol/g substitution, 15 mmol) of the resin in 200mL of a 1:1 mixture of dimethylformamide and dichloromethane for 45minutes, followed by filtering and washing with 2×125 mL ofdimethylformamide. The washed resin was then deprotected with 2×125 mLof 20% piperidine in dimethylformamide for 15 minutes, filtered, andwashed with 4×125 mL of dimethylformamide.

A solution of 21.5 g (MW=717, 30 mmol) of Fmoc-protected surrogate (7)in 160 mL of dimethylformamide was added to the deprotected Sieber amideresin as prepared above, followed by 15.6 g (MW=520.3, 30 mmol) of solidPyBop, and 10.4 mL (MW=129.25, d=0.742, 60 mmol) ofdiisopropylethylamine, followed by another 40 mL of dimethylformamide.The mixture was agitated overnight with nitrogen bubbling. The resin wasfiltered, and washed with 4×130 mL of dimethylformamide, capped with 150mL of capping solution consisting of a 3:2:1 solution ofdimethylformamide:acetic anhydride:pyridine for 30 minutes, filtered,and washed with 4×130 mL of dimethylformamide to provide surrogate (7)complexed to resin.

The resulting Fmoc-protected surrogate (7) complexed to resin wasdeprotected with 2×130 mL of 20% piperidine in dimethylformamide for 15minutes, filtered, and washed with 4×130 mL of dimethylformamide toyield surrogate (7) complexed to resin. A solution of 27.6 g ofFmoc-Tyr-(tBu)-OH (60 mmol, 4 eq.) in dimethylformamide (200 mL) wasadded to surrogate (7) complexed to resin, followed by a solution of24.8 g of HCTU (60 mmol, 4 eq.), and 20.8 mL (120 mmol, 8 eq.) ofdiisopropylethylamine in DMF to a final volume of 200 mL and coupledovernight with nitrogen bubbling. The resulting Fmoc-Tyr-(tBu)-surrogate(7)-resin was isolated by filtration and washed with 2×130 mL ofdimethylformamide. In order to ensure complete coupling, the product wasagain treated with a solution of 27.6 g of Fmoc-Tyr-(tBu)-OH (MW=459.6,60 mmol, 4 eq.) in dimethylformamide to a final volume of 200 mLfollowed by a solution of 24.8 g of HCTU (60 mmol, 4 eq.), anddiisopropylethylamine (20.8 mL, 120 mmol, 8 eq.) in DMF to a finalvolume of 200 mL and coupled overnight with nitrogen bubbling. The resinwas filtered, and washed with 2×130 mL of dimethylformamide. HPLC andLC/MS showed that coupling between surrogate (7)-resin andFmoc-Tyr-(tBu)-OH was complete.

The resulting Fmoc-Tyr-(tBu)-surrogate (7)-resin was then capped with150 mL of capping solution as above for 30 minutes. The resin was thenfiltered, washed with 4×130 mL of dimethylformamide, 4×130 mL ofdichloromethane, 2×130 mL of MeOH, 2×130 mL of diethyl ether, and driedunder vacuum to give 36.7 g.

Thereafter each succeeding amino acid may be coupled. Before thecoupling of the first amino acid, resulting Fmoc-Tyr-(tBu)-surrogate(7)-resin was swollen for 45 minutes with 200 mL of a 1:1 solution ofdimethylformamide:dichloromethane. Each amino acid (Fmoc-AA-OH) wascoupled by repeating the following cycle. The terminal amino acidresidue was deprotected with 2×125 mL of 20% piperidine indimethylformamide for 15 minutes, filtered and washed with 4×125 mL ofdimethylformamide. The beads were checked by ninhydrin test. A solutionof Fmoc-AA-OH (60 mmol, 4 eq.) in dimethylformamide to a final volume of200 mL was added to resin, followed by a solution of HBTU (60 mmol, 4eq.), and (120 mmol, 8 eq.) of N-methylmorpholine in DMF to a finalvolume of 200 mL [concentration of Fmoc-AA-OH=150 mM solution] andcoupled for 30 minutes with nitrogen bubbling (coupling reaction checkedby ninhydrin test). When the ninhydrin test was negative, the resin wasfiltered, and washed with 4×130 mL of dimethylformamide.

After all amino acids had been coupled, the resin was washed with 4×130mL of dichloromethane, 4×130 mL of methanol, 4×130 mL of diethyl ether,and dried under vacuum to give product. The weight increase wasquantitative.

100 mL of cleavage reagent consisting of a 81.5:5:5:5:2.5:1 solution oftrifluoroacetic acid:phenol:thioanisole:water:DDT:triisopropyl silanewas added to 32 g (˜6.4 mmol) of the following linear construct:

The suspension was allowed to stand at room temperature for 5 minutesand then filtered. Another 100 mL of cleavage reagent was added to theresin, allowed to stand for 5 minutes, and filtered. This process wasrepeated.

The resulting resin was then washed with 2×40 mL of trifluoroaceticacid. The filtrates were combined and stirred for 2.5 hours at roomtemperature, and then concentrated under reduced pressure to ˜100 mLvolume. Cold diethyl ether (1.5 L, pre-cooled to −20° C.) was added tothe filtrate, and then placed in the freezer (−20° C.) for 1 hour,filtered through a sintered glass funnel, and the solids washed with3×200 mL of cold diethyl ether, and then dried under vacuum for 1 hourwith the solids triturated every 15 minutes to make sure solvent wasremoved efficiently. The following construct was obtained (15.4 g) (103%overall crude yield):

The above construct (15.4 g, 6.4 mmol) was dissolved in 16 L of 30%acetonitrile in water. The pH was adjusted to 8.4 using a solution of 5%ammonium hydroxide. Pulverized activated carbon (15.4 g) was added, andthe suspension stirred overnight. The carbon was removed by filtrationthrough celite. The celite was washed 3×100 mL 50% acetonitrile inwater. The filtrates were combined, diluted with water to a finalconcentration of 10% acetonitrile, and loaded in the column forpurification. Purification of the trifluoroacetate salt of the resultingconstruct was performed under the following conditions:

Column: Luna C₁₈, 10 μ, 50×33 mm

Flow: 70 mL/minute

Solvent A: water containing 0.1% trifluoroacetic acid

Solvent B: acetonitrile containing 0.1% trifluoroacetic acid

Gradient: 5% solvent B for 5 minutes 26% B to 52% B in 30 minutes

The pure fractions were combined and lyophilized to give the purifiedtrifluoroacetate salt of the construct. Dowex SBR, LCNG-OH resin (450 g)was suspended in 2 L of water, and gently stirred for 15 minutes,allowed to stand for 15 minutes, and then decanted. The procedure wasrepeated, and then 0.5 L of water added, and the slurry transferred intoa 6×60 cm column. The water was drained, washed with 4 L of water, andions exchanged with 6.5 L of 20% acetic acid solution. The resin wasallowed to stand at room temperature overnight, and then washed withwater until the pH of the filtrate was ˜4 (8 L of water used). Thetrifluoroacetate salt of the above construct (11.1 g), as preparedabove, was dissolved in 80 mL of water, and loaded to the ion exchangeresin, and eluted with water. Fractions containing 79-1 were combined,and 20% acetic acid solution was added to adjust the final concentrationto 5% acetic acid, and then lyophilized. The following construct 1-132(10.4 g) was obtained:

Similar methods may be employed with any construct where the surrogateis bound to resin or other peptide solid support and is at theC-terminal position.

Optional PEGylation of the peptide constructs of the invention may beperformed in any manner, such as those described below.

PEGylation of reactive amine groups, such as lysine or ornithine sidechains, an omega amino aliphatic in position Aaa¹, or an amine group inJ of an amino acid surrogate at Aaa¹⁵ was accomplished by dissolving0.005 mmol purified construct in 2 mL of dimethylsulfoxide, followed bythe addition of 55.5 mg (0.011 mmol, 2 eq) of PEG-5K-OSu (5,000 Da MWmethoxy-PEG with a succinimidyl propionate reactive group), with 17.7 μL(0.13 mmol, 20 eq.) of triethyl amine then added, and the slightlycloudy solution stirred at room temperature for 3 hours. ExcessPEG-5K-OSu was quenched by the addition of 7 μL (0.111 mmol, 10 eq.) ofethanol amine, and the reaction stirred overnight.

PEGylation of reactive carboxyl groups, such as Asp or Glu side chainsor a terminal carboxyl at Aaa¹⁵ on either a residue or surrogate, isaccomplished by coupling PEG-NH₂ (PEG-amine), to the constructcontaining a carboxylate group in the side chain of Asp or Glu or at theC-terminus. The peptide construct (0.005 mmol) is dissolved in DMSO (2mL), followed by the addition of 55.5 mg (0.011 mmol, 2 eq) of PEG-NH₂and HOBt (0.01 mmol). The coupling is started by the addition of 0.0055mmole of coupling reagentN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide (EDAC). The slightlycloudy solution stirred at room temperature overnight. The PEGylatedpeptide construct is then purified by HPLC.

PEGylation of reactive thiol groups, such as Cys or Hcys side chains ora thiol group in Q of an amino acid surrogate at Aaa¹, is accomplishedby treating the peptide construct in DMSO with PEG-methyl-maleimidereagent (SunBio, Orinda, Calif.) overnight. The PEGylated peptideconstruct is then purified by HPLC.

Following PEGylation, the resulting crude mixture was then purified byHPLC, yielding a PEG derivatized construct including one or more aminoacid surrogates.

In Vitro and In Vivo Test Systems

Selected constructs were tested in assays to determine binding andfunctional status. The following assays were employed.

Cell culture. A cDNA clone that encodes for human natriuratic peptidereceptor A (NPRA) was purchased from Bio S&T Inc. (Montreal, Quebec).The cDNA clone was inserted into the mammalian expression vectorpcDNA3.1 (Invitrogen) and transfected into HEK-293 cells. Stable cloneswere selected by culture of cells in the presence of G418 sulfate.Expression of NPRA was examined by binding of [¹²⁵I]-atrial natriureticpeptide ([¹²⁵I]-ANP) to membrane homogenates prepared from clonal celllines. HEK-hNPRA cells were maintained in culture at 37° C. in 5% CO₂ inDulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS,G418 sulfate (300 μg/mL) sodium glutamate (0.29 mg/mL), penicillin (100units/mL) and streptromycin (100 ug/mL).

Competitive binding assay. A competitive inhibition binding assay wasperformed using crude membrane homogenates prepared from HEK-hNPRAcells. To prepare membrane homogenates, cells were rinsed withphosphate-buffered saline and incubated for 15 minutes at 4° C. inhypotonic lysis buffer (10 mM Tris, pH 7.4+5 mM EDTA). Cells weretransferred from plates to polypropylene tubes and homogenized.Homogenates were centrifuged at 25,000×g for 20 minutes. Pellets wereresuspended in buffer consisting of 50 mM Tris (pH 7.4) and 1 mM EDTA,homogenized and centrifuged at 25,000×g for 20 minutes. Pellets wereresuspended in buffer consisting of 100 mM Tris (pH 7.4) and 10 mM MgCl₂and stored at −80° C. until needed. On the day of an assay, homogenateswere thawed and homogenized. Binding of [¹²⁵I]-ANP was carried out inbuffer containing 25 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM CaCl₂, 5 mMMgCl₂, 0.1% BSA and 1 mM 1,10-phenanthroline. Homogenates (1-10 μgprotein/well) were incubated with [¹²⁵I]-ANP (25-30 pM) and increasingconcentrations of competing ligands in Millipore filter plates for 120minutes at 4° C. Assays were stopped by addition of cold wash buffer(phosphate-buffered saline) followed by filtration using a vacuummanifold. Bound radioactivity was determined using a gamma counter.Non-specific binding was defined by binding of [I¹²⁵]-hANP tonon-transfected HEK293 membranes. Data were analyzed using GraphPadPrism® curve-fitting software.

General method for determination of EC₅₀. Functional evaluation ofconstructs was performed by measuring the accumulation of intracellularcGMP in HEK-293 cells that express recombinant hNPR-A. HEK-NPRA cellswere harvested by washing and centrifugation in Cell Dissociation Buffer(Gibco, Life Technologies). Pelleted cells were resuspended in Hank'sBalanced Salt Solution (HBSS) containing 10 mM Hepes (pH 7.4), 5 mMMgCl₂, 200 mM L-glutamine, 1 mM 1,10-phenanthroline and BSA (0.5 mg/mL).Following centrifugation, cells were resuspended in the above buffersupplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX). Cells(˜2×10⁵/well) were added to each well of a 96-well plate and incubatedfor 15 minutes at 37° C. Following the pre-incubation period, cells wereincubated for an additional 15 minutes in the presence of increasingconcentrations of constructs. The reaction was terminated by lysis ofthe cells with temperature shock. The reaction plate was incubated in adry ice/ethanol bath for 15 minutes followed by incubation at 90° C. for10 minutes. Accumulation of cGMP was measured using the cGMP FlashplateRIA (Perkin-Elmer). Data analysis and EC₅₀ values were determined byusing nonlinear regression analysis with GraphPad Prism® software.

Determination of mass and nuclear magnetic resonance analysis. The massvalues of PEG-conjugated constructs were analyzed by MALDI-TOF massspectrometry (positive ion mode) using alpha-cyano-4-hydroxycinnamicacid (CHCA) as matrix. Methanol was used for sample preparation inconstruct to matrix ratios of 1:10, 1:20 and 1:30. Alternatively othermatrices such as, sinapinic acid (SA) and 2, 5-dihydroxybenzoic acid(DHB), and solvents such acetonitrile—0.1% aqueous TFA can be used forsample preparation. Other determinations of mass values were made usinga Waters MicroMass ZQ device utilizing a positive mode. For constructsthat were not PEGylated, mass determinations were compared withcalculated values and expressed in the form of mass weight plus twodivided by two ((M+2)/2), unless otherwise specified.

Proton NMR data was obtained using a Bruker 300 MHz spectrometer. Thespectra were obtained after dissolving constructs in a deuteriatedsolvent such as chloroform, DMSO, or methanol as appropriate.

HPLC measurements were made using a Waters Alliance HT with a YMC PackPro C₁₈ column (4.6×50 mm, 3μ) eluted at 1 mL/minute in a step-wiseprocedure. Solvent A (water containing 0.1% trifluoroacetic acid v/v)and solvent B (acetonitrile containing 0.1% trifluoroacetic acid v/v)were used as mobile phases. For analysis of keto piperazineintermediates, the column was equilibrated with 10% B and then B wasincreased to 90% over a period of 8 minutes. For analysis of peptides,the column was equilibrated with 2% B and then B was increased to 90%over a period of 8 minutes.

Animal Models—Blood Pressure Transducer Implantation. Rats are inducedto a surgical plane of anesthesia with isoflurane and maintained on aheating pad. The abdomen is shaved and scrubbed with 70% alcohol andbetadine solution. Using aseptic technique, a midline abdominal incisionis made in order to expose the descending aorta and vena cava. Thecontents of the abdomen are retracted gently using wet sterile gauze andretractors. Based on the manufacturer's instructions (described in DataSciences International's Multiplus TL Series Device Surgical Manual2000: pp. 3.1-3.10), the abdominal aorta is carefully dissected from thesurrounding fat and connective tissue and the catheter of the bloodpressure transducer is inserted. The catheter of the transducer issecured into place using surgical glue and the body of the transducerstabilized by suturing to the abdominal wall (4-0 silk suture). Care istaken to ensure that hemostasis is maintained during the procedure andthat blood flow is not compromised (e.g. aorta will not be occluded formore than 3 minutes at a time). Transducer placement is verified usingthe telemetry radio signal. After transducer placement, the gauzesponges are removed and the abdominal cavity is flushed with sterilesaline. The abdominal incision is then sutured closed with nonabsorbablesutures (4-0 silk suture) in a simple interrupted pattern. The skin isclosed using absorbable suture (4-0 vicryl). Finally the animal isremoved from the isoflurane and placed in a warm environment while beingmonitored until it is fully awake.

Surgical Induction of Congestive Heart Failure (Volume Overload). Inthis procedure, the descending aorta and vena cava are exposed in thesame manner as it is in the implantation procedure for the telemetrydevice. Once access to the vessels between the renal and iliacbifurcation is obtained, a puncture is made with a 1.8 mm needle(outside diameter) to the descending aorta. The needle is advanced intothe inferior vena cava and withdrawn. The ventral puncture site in thedescending aorta is sealed with tissue adhesive. The persistence of ashunt between the aorta and vena cava is confirmed visually by theswelling of the vena cava and the mixing of the venous and arterialblood. In the event that a pressure transducer is also implanted, thetwo procedures are done concurrently. The general methods described inFlaim, S. F., W. J. Minteer, S. H. Nellis, and D. P. Clark: Chronicarteriovenous shunt: evaluation of a model for heart failure in rat. Am.J. Physiol. 236:H698-H704 (1979) and Garcia, R. and S. Diebold: Simple,rapid and effective method of producing aortocaval shunts in the rat.Cardiovasc. Res. 24:430-432 (1990), are incorporated here by reference.

Blood Pressure Monitoring. Telemetry signals from the blood pressuretransducers (model TA11PA-C40, Data Sciences International, St Paul,Minn.) are collected and analyzed using Dataquest A.R.T. Gold softwareversion 3.0 (Data Sciences International). Rats were observed atapproximately the same time each day. Each rat, in its home cage, isplaced on a receiver in the observation room and allowed to adjust tothe change in location for 30 minutes. Baseline recordings are taken for30 minutes just prior to dosing and treatment recordings are taken for135 minutes immediately following IV dosing and 210 minutes following SCdosing. The data are compared to the results after saline dosing in away similar to methods previously published in Clemens, L. E., R. G.Almirez, K. A. Baudouin, E. B. Grossbard, and A. A. Protter: Human brainnatriuretic peptide reduces blood pressure in normotensive and acutenorepinephrine-induced hypertensive rabbits. Am. J. Hypertens.10:654-661 (1997), incorporated here by reference.

Diuresis and Natriuresis. Rats are induced to a surgical plane ofanesthesia with sodium pentobarbital and maintained on a heating pad.The abdomen is shaved and scrubbed with 70% alcohol and betadinesolution. Using aseptic technique, a midline abdominal incision is madein order to expose the urinary bladder. A purse-string suture isintroduced to ventral surface of the bladder and a small incision ismade within the suture area. The flared end of a catheter in insertedinto the open in and the purse-string suture tightened around it tosecure it in place. Urine is collected into preweighed microcentrifugetubes at various time intervals before and after dosing in a way similarto methods published in Abassi, Z. A., J. R. Powell, E. Golomb, and H.R. Keiser: Renal and systemic effects of urodilatin in rats withhigh-output heart failure. Am. J. Physiol. 262:F615-F621 (1992),incorporated here by reference. Urine volume is measured by weight.

EXAMPLE 1

The following constructs were synthesized, using amino acid surrogatesof one or more of the foregoing methods, were purified and the massweights determined, with the results as shown below:

TABLE 1 Number (M + 2)/2 Structure 1-1  931.9

1-2  932.0

1-3  938.9

1-4  932.0

1-5  932.9

1-6  932.7

1-7  932.3

1-8  932.0

1-9  932.2

1-10  931.6

1-11  931.6

1-12  931.6

1-13  931.3

1-14  931.6

1-15  966.4

1-16  966.4

1-17  966.0

1-18  966.4

1-19  965.7

1-20  965.9

1-21  965.7

1-22  965.6

1-23 1000.4

1-24  938.6

1-25  931.6

1-26  938.2

1-27  962.2

1-28  965.9

1-29  932.8

1-30  927.8

1-31  972.8

1-32  972.9

1-33  923.2

1-34  944.7

1-35  943.7

1-36  923.0

1-37  944.6

1-38  957.7

1-39  965.6

1-40  965.8

1-41  931.3

1-42  965.4

1-43  965.4

1-44  965.5

1-45  965.3

1-46  965.3

1-47  951.3

1-48  951.2

1-49  958.2

1-50  965.2

1-51  965.3

1-52  919.6

1-53  965.6

1-54  944.6

1-55  944.6

1-56  930.9

1-57  924.0

1-58  931.0

1-59  938.1

1-60  896.0

1-61  924.1

1-62  987.7

1-63  931.1

1-64  931.1

1-65  931.2

1-66  938.0

1-67 1000.1

1-68  973.1

1-69  924.9

1-70  853.5

1-71  931.2

1-72  874.4

1-73  944.4

1-74  952.1

1-75  930.9

1-76 1007.1

1-77  992.4

1-78  958.7

1-79  965.6

1-80  958.8

1-81  964.8

1-82  948.9

1-83  972.7

1-84  965.9

1-85  993.2

1-86 1000.9

1-87  993.7

1-88  966.7

1-89  966.9

1-90  966.6

1-91  967.0

1-92  966.9

1-93  966.7

1-94  966.9

1-95  966.7

1-96  966.7

1-97  966.7

1-98  966.7

1-99  966.5

1-100  881.8

1-101  922.4

1-102  938.0

1-103  937.8

1-104  931.1

1-105  931.2

1-106  930.9

1-107  938.5

1-108  931.0

1-109  945.2

1-110  938.1

1-111  931.5

1-112 1929.6 (M + 1)

1-113  965.2

1-114  965.3

1-115 1822.3 (M + 1)

1-116 1849.3 (M + 1)

1-117  978.7

1-118  965.0

1-119  937.0

1-120  960.5

1-121  967.0

1-122  981.0

1-123  953.4

1-124  922.8

1-125  965.2

1-126  967.3

1-127  967.3

1-128 1030.3

1-129  995.7

1-130  939.1

1-131  932.0

1-132  938.7

1-133  974.0

1-134  949

1-135  966.3

1-136 1022.6

1-137 1015.5

1-138  980.7

1-139  930.8

1-140  923.8

1-141  902.9

1-142  988.1

1-143 1023.4

1-144 1033.6

1-145 1001.5

1-146  888.9

1-147 1001.6

1-148  910.5

1-149  967.1

1-150  966.0

1-151  973.1

1-152  973.1

1-153  923.5

1-154  938.1

1-155  938.8

1-156  930.9

1-157  931.2

1-158  931.3

1-159  931.3

1-160  931.3

1-161  931.1

1-162  931.3

1-163  930.5

1-164  938.3

1-165  930.8

1-166  945.1

1-167  930.9

1-168  931.1

1-169  938.0

1-170  931.1

1-171  930.4

1-172  930.5

1-173  937.7

1-174  937.2

1-175  930.4

1-176  916.5

1-177  916.4

1-178  944.6

1-179  937.1

1-180  944.6

1-181  930.4

1-182  930.3

1-183  930.3

1-184  930.9

1-185  930.4

1-186  930.4

1-187  937.7

1-188  938.8

1-189  937.3

1-190  937.6

1-191  938.5

1-192  938.4

1-193  938.9

1-194  938.3

1-195  931.5

1-196  931.4

1-197  938.7

1-198  931.4

1-199  938.3

1-200  931.3

1-201  931.6

1-202  938.5

1-203  938.2

1-204  938.4

1-205  937.9

1-206  930.7

1-207  930.7

1-208  937.7

1-209  930.8

1-210  930.5

1-211  930.5

1-212  930.8

1-213  931.3

1-214  944.9

1-215  937.9

1-216  930.6

1-217  930.7

1-218  930.8

1-219  930.8

1-220  930.9

1-221  931.6

1-222  937.8

1-223  931.1

1-224  938.2

1-225  937.8

1-226  945.4

1-227  938.5

1-228  930.5

1-229  930.4

1-230  930.5

1-231  930.9

1-232  938.8

1-233  930.8

1-234 1030.8

1-235 1023.3

1-236 1031.2

1-237  938.1

1-238  971.8

1-239  955.4

1-240  917.4

1-241  937.8

1-242  938.5

1-243  930.8

1-244  930.8

1-245  963.3

1-246  944.8

1-247  959.9

1-248  964.3

EXAMPLE 2

The following constructs of Table 2 are synthesized, using amino acidsurrogates of one or more of the foregoing methods, are purified and themass weights determined:

TABLE 2 Num- ber Structure 2-1

2-2

2-3

2-4

2-5

2-6

2-7

2-8

2-9

2-10

2-11

2-12

2-13

2-14

2-15

2-16

2-17

2-18

2-19

2-20

2-21

2-22

2-23

2-24

2-25

2-26

2-27

EXAMPLE 3

Construct 1-1 (SEQ ID NO:3), with the following structure, was tested asdescribed above.

In receptor binding studies this construct had an average Ki of 0.3 nMin an assay system in which hANP had a Ki of 0.05 nM and mini-ANP had aKi of 0.6 nM. Construct 1-1 had an EC₅₀ of 2 nM in an assay system inwhich hANP had an EC₅₀ of 0.6 nM and mini-ANP had an EC₅₀ of 3.3 nM.

EXAMPLE 4

Construct 1-9 (SEQ ID NO:14), (SEQ ID NO:15), with the followingstructure, was tested as described above.

In receptor binding studies this construct had an average Ki of 0.9 nMin an assay system in which hANP had a Ki of 0.05 nM and mini-ANP had aKi of 0.6 nM. Construct 1-9 had an EC₅₀ of 3.5 nM in an assay system inwhich hANP had an EC₅₀ of 0.6 nM and mini-ANP had an EC₅₀ of 3.3 nM.

EXAMPLE 5

Construct 1-8 (SEQ ID NO:13), with the following structure, was testedas described above.

In receptor binding studies this construct had an average Ki of 0.2 nMin an assay system in which hANP had a Ki of 0.05 nM and mini-ANP had aKi of 0.6 nM. Construct 1-8 had an EC₅₀ of 2 nM in an assay system inwhich the construct of FIG. 1 had an EC₅₀ of 0.6 nM and mini-ANP had anEC₅₀ of 3.3 nM.

EXAMPLE 6

Construct 1-18 (SEQ ID NO:20), with the following structure, was testedas described above.

In receptor binding studies this construct had an average Ki of 0.027 nMin an assay system in which hANP had a Ki of 0.05 nM and mini-ANP had aKi of 0.6 nM. Construct 1-18 had an EC₅₀ of 0.2 nM in an assay system inwhich hANP had an EC₅₀ of 0.6 nM and mini-ANP had an EC₅₀ of 3.3 nM.

Construct 1-18 was stable in both rat and human plasma, with T_(1/2) of≈2 hours at 37° C. When administered IV, the in vivo T_(1/2) in rats was˜20 minutes. Approximately 25 to 50% of the injected dose wasbioavailable in rats when administered by a subcutaneous route. FIG. 2depicts the concentration of construct 1-18 in ng/mL over time in rats,with the curve for “SC” indicating subcutaneous administration at a doseof 5 mg/kg, and the curve for “IV” indicating intravenous administrationat a dose of 2 mg/kg.

EXAMPLE 7

Blood pressure transducers were implanted in rats as described under theheading “Animal Models—Blood Pressure Transducer Implantation.” Studieswere conducted as described under the heading “Blood PressureMonitoring.” Studies included determination of changes in systolic bloodpressure compared to saline following administration of constructs ofthe invention. In one study, rats were administered construct 1-63 (SEQID NO:20) by an IV route at 0.03 mg/kg body weight (n=4), 0.1 mg/kg(n=7) or 0.3 mg/kg (n=8). The blood pressure was monitored at 5 minutesprior to IV administration and at 5, 10 and 15 minutes afteradministration, and thereafter at 15 minute intervals until 135 minutespost administration. At all time points for all doses the measuredsystolic blood pressure was lower than saline control, with the decreasein blood pressure ranging from a minimum of about 5% to a maximum ofabout 19%, generally in a dose dependent manner, and with the greatestresponse seen at 10 to 45 minutes after administration.

In a second study, rats were administered construct 1-63 (SEQ ID NO:20)by a subcutaneous route at 0.3 mg/kg body weight (n=8), 1.0 mg/kg (n=7)or 3.0 mg/kg (n=7). The blood pressure was monitored at 15 and 5 minutesprior to SC administration and at 5, 10 and 15 minutes afteradministration, and thereafter at 15 minute intervals until 210 minutespost administration. At all time points for all doses the measuredsystolic blood pressure was lower than saline control. At 0.3 mg/kg SC,the decrease in systolic pressure was in the range of 2% at time pointsafter 2 hours post administration, which was within the error range.However, at all other time points for 0.3 mg/kg SC, and at all timepoints for 1.0 and 3.0 mg/kg, the decrease was statistically differentand lower than the saline control. At 3.0 mg/kg a maximum decrease ofapproximately 20% to 23% in systolic pressure was seen at 45 to 120minutes post administration, with a maximum decrease in the same timeperiod at 1.0 mg/kg of 17% to 19%.

In a third study study, rats were administered construct 1-18 (SEQ IDNO:20) by an IV route at 0.3 mg/kg of body weight (n=8). The bloodpressure was monitored at 5 minutes prior to IV administration and at 5,10 and 15 minutes after administration, and thereafter at 15 minuteintervals until 135 minutes post administration. At all time points forall doses the measured systolic blood pressure was lower than salinecontrol, with the decrease in blood pressure ranging from a minimum ofabout 5% to a maximum of about 13%, with the greatest response seen at15 minutes after administration.

In a fourth study, rats were administered construct 1-18 (SEQ ID NO:20)by a subcutaneous route at 0.1 mg/kg body weight (n=4), 0.3 mg/kg (n=7)or 1.0 mg/kg (n=8). The blood pressure was monitored at 15 and 5 minutesprior to SC administration and at 5, 10 and 15 minutes afteradministration, and thereafter at 15 minute intervals until 225 minutespost administration. At all time points for all doses the measuredsystolic blood pressure was lower than saline control. At 0.1 mg/kg SC,the decrease in systolic pressure was not statistically relevant afterabout two and one-half hours. However, at all other time points for 0.1mg/kg SC, and at all time points less than about two and one-half hoursfor 0.3 and at all time points for 1.0 mg/kg, the decrease wasstatistically different and lower than the saline control. At 1.0 mg/kga maximum decrease of approximately 9% to 13% in systolic pressure wasseen at 45 to 120 minutes post administration.

EXAMPLE 8

Total urine output in rats was measured as described under the heading“Diuresis and Natriuresis.” Groups of four animals were administeredconstructs 1-18 (SEQ ID NO:20) and 1-63 (SEQ ID NO:44) by IV routes,with animals receiving 0.03 mg/kg of body weight, 0.1 mg/kg and 0.3mg/kg of construct 1-18, and 0.1 mg/kg of body weight, 0.3 mg/kg and 1.0mg/kg of construct 1-63(SEQ ID NO:44). Total urine output over 30minutes postdose was measured, with results as shown in FIG. 3.

In a separate study, total urine about was similarly measured in a groupof four animals receiving doses of 0.3, 1.0 and 3.0 mg/kg of body weightof construct 1-63 (SEQ ID NO:44) by a SC route, with saline used as acontrol. Total urine output over 45 minutes postdose was measured.Results are as shown in FIG. 4.

EXAMPLE 9

The pharmacokinetics of selected constructs of the invention werestudied in male Sprague-Dawley rats following intravenous (IV) orsubcutaneous (SC) administration. Pharmacokinetic parameters of selectedconstructs in rats were determined and summarized in Tables 3 and 4.

Constructs of the invention as indicated in Tables 4 and 5 were preparedas the TFA salt and dissolved in saline at 1 mL/kg for both IV and SCdosing routes. The IV dose was administered via a femoral artery cannulaat target doses of 0.3 and 2 mg/kg. The SC dose was administered attarget doses of 1 and 5 mg/kg. Animals were not fasted overnight beforedosing. Blood was collected into containers containing dipotassium EDTAat predetermined intervals from a previously implanted cannula in thejugular vein. Plasma was obtained by centrifugation of the blood andstored at −70° C. until analysis.

Data analysis utilized a LC-MS/MS system including a Leap TechnologiesHTS-PAL autosampler equipped with a 100 μL injection loop, two ShimadzuPumps and a Sciex API 4000 mass spectrometer. Chromatographic separationof the analytes was achieved on a Luna C₁₈ column (4.6×100 mm; 3μ)eluted at 1 mL/min with a step-wise procedure. Solvent A (watercontaining 0.1% formic acid v/v) and solvent B (acetonitrile containing0.1% formic acid v/v) were used as the mobile phases. Initially, thecolumn was equilibrated with 5% B and, 2 minutes after sample injection,B was increased to 60% over a period of 0.5 minutes and held at thisconcentration for 1.1 minutes. The composition of B was increased to 80%in 1.4 minutes and maintained for 0.3 minutes. The composition of B wasreturned to 5% in 0.2 minutes. The total run time was 6 minutes. Massspectrometric detection of the analytes was accomplished using the TurboIonspray interface operated in the positive ion mode. Analyte responsewas measured by multiple reaction monitoring (MRM) of the transitions ofthe protonated precursor ions to the selected product ions.

Aliquots of plasma (100 μL) were mixed with the internal standard (IS)and subjected to solid phase extraction using C₈ cartridges in a 96 wellformat. After preconditioning of the C₈ cartridges with 1 mL of methanoland 1 mL of 2% ammonium hydroxide in water, plasma samples were loadedonto the cartridges. Following washing the cartridges with 2% ammoniumhydroxide in 40% methanol, constructs were eluted from the cartridgeswith 1 mL of 2% acetic acid in 60% methanol. The eluents weretransferred to a clean plate, evaporated under a stream of N₂ and theresidues were resuspended in 100 μL of 20 mM ammonium acetate andacetonitrile (6:4, v:v) prior to LC-MS/MS analysis. Calibrationstandards (2-1000 ng/mL) were prepared in the same manner by addingconstruct at various concentrations and its respective IS to 100 μL ofuntreated rat plasma. Similarly, quality control samples were preparedby adding construct and IS to 100 μL of control plasma at 3 differentconcentrations (3.5, 75 and 750 ng/mL) of construct.

Data were acquired and processed by Sciex Analyst 1.4.1 software. Peakarea ratios of construct to IS were plotted as a function of the nominalconcentrations of construct. Linear regression using a weighting factorof 1/x was used to calculate the concentration of construct in plasmasamples. The lower limit of quantification (LLOQ) for this assaytypically was 2 or 5 ng/mL.

Pharmacokinetic parameters were calculated by establishednon-compartmental method (Win-Nonlin version 2.1; Consulting Inc., PaloAlto, Calif.). The area under the plasma concentration versus time curve(AUC) was determined using linear trapezoidal interpolation in theascending slope and logarithmic trapezoidal interpolation in thedescending slope. The portion of the AUC from the last measurableconcentration to infinity was estimated from the equation C_(t)/k_(el),where C_(t) represents the last measurable concentration and k_(el) isthe elimination rate constant. The latter was determined from theconcentration versus time curve by linear regression at the terminalphase of the semi-logarithmic plot.

TABLE 3 Summary of Pharmacokinetic Parameters of Constructs in SD RatsFollowing IV Administration PK Parameters Dose AUC Vdss Cl (mL/ T_(1/2)Construct (mg/kg) (nM · hr) (L/kg) min/kg) (hr) 1-18 (SEQ ID NO: 20) 2263 0.7 72 0.2 1-18 (SEQ ID NO: 20) 0.3 61 0.4 43 0.2 1-63 (SEQ ID NO:20) 2 67 4.6 290 0.3 1-63 (SEQ ID NO: 20) 0.3 33 5.8 546 0.3 1-55 (SEQID NO: 39) 2 828 0.3 21 0.3 1-55 (SEQ ID NO: 39) 0.3 116 0.2 15 0.2 1-54(SEQ ID NO: 38) 2 277 1.0 66 0.2 1-35 (SEQ ID NO: 31) 2 10 57 1857 0.41-39 2 142 2.8 134 0.3 1-42 2 369 0.7 50 0.3 1-45 2 850 0.3 23 0.3 1-282 351 1.1 51 0.4 1-69 (SEQ ID NO: 43) 2 113 0.8 168 0.1 1-100 (SEQ IDNO: 29) 2 42 0.8 456 0.1 1-68 2 478 0.5 40 0.3 1-43 2 1247 0.6 14 0.71-44 2 1539 0.4 15 0.5 1-51 (SEQ ID NO: 20) 2 190 1.0 94 0.2

TABLE 4 Pharmacokinetics of Constructs in SD Rats Following SCAdministration PK Parameters in SD Rats Dose AUC Tmax Cmax T_(1/2)Construct (mg/kg) (nM · hr) (hr) (nM) (hr) 1-18 (SEQ ID NO: 20) 5 2550.2 131 1.6 1-18 (SEQ ID NO: 20) 1 40 0.1 85 0.2 1-55 (SEQ ID NO: 39) 5232 0.3 250 0.5 1-63 (SEQ ID NO: 20) 1 7 0.1 26 0.2

EXAMPLE 10

A formulation of 1-132 was made for pharmaceutical use. 1-132 was usedin the acetate salt form. The formulation was dispensed into a 1 mL vialwhich was stoppered and sealed, with each vial containing:

-   -   0.1 mg of 1-132 acetate, based on peptide weight net of acetate    -   1.181 mg succinic acid, NF    -   47.0 mannitol, USP    -   1N NaOH, USP, as needed to adjust pH    -   1N HCl, USP, as needed to adjust pH    -   Water for injection, to 1 mL volume        The pH of the final product was adjusted to pH 4.00±0.05 with 1N        NAOH or 1N HCl, as required. The resulting solution was filtered        through a sterile 0.22 micron filter prior to vialing, and was        stored at 5° C. until used.

An alternative formulation of 1-132 was made for pharmaceutical use,similar to the formulation above, but additionally including betweenabout 0.02 mg and 0.06 mg of disodium pamoate, such that the resultingsolution was a pamoate suspension.

EXAMPLE 11

The following constructs were synthesized, using amino acid surrogatesof one or more of the foregoing methods, purified and conjugated withPEG-5K-OSu and the mass weights determined, with the results as shown inTable 5 below:

TABLE 5 Construct (M + 1) Structure 5-1 6391-8332

5-2 6338-8412

5-3 6427-8159

5-4 6406-8219

5-5 11959- 13514

5-6 6602-8279

5-7 6506-8580

5-8 6319-8387

5-9 6660-8505

5-10 6444-8174

5-11 6383-8103

5-12 6479-8289

5-13 6564-8869

EXAMPLE 12

The following constructs of Table 6 are synthesized, using amino acidsurrogates of one or more of the foregoing methods, purified andconjugated with PEG-5K-OSu or another reactive PEG, and the mass weightsdetermined:

TABLE 6 Con- struct Structure 6-1

6-2

6-3

6-4

6-5

6-6

6-7

6-8

6-9

6-10

6-11

6-12

EXAMPLE 13

Construct 5-1 (SEQ ID NO:83), with the following structure, was testedas described above.

In receptor binding studies this construct had an average Ki of 70 nM inan assay system in which hANP had a Ki of 0.05 nM and mini-ANP had a Kiof 0.6 nM.

EXAMPLE 14

Construct 5-9 (SEQ ID NO:20), with the following structure, was testedas described above.

In receptor binding studies this construct had an average Ki ofapproximately 2 nM in an assay system in which hANP had a Ki of 0.05 nMand mini-ANP had a Ki of 0.6 nM. Construct 5-9 had an EC₅₀ ofapproximately 9.5 nM in an assay system in which hANP had an EC₅₀ of 0.6nM and mini-ANP had an EC₅₀ of 3.3 nM.

EXAMPLE 15

Any of constructs of the invention, including without limitationconstructs 1-1 to 1-248, 2-1 to 2-21, 5-1 to 5-18 and 6-1 to 6-12, isformulated for time-release injection. Any of the constructs isformulated with a PEG, such as poly(ethylene glycol) 3350, andoptionally one or more additional excipients and preservatives,including but not limited to excipients such as salts, polysorbate 80,sodium hydroxide or hydrochloric acid to adjust pH, and the like.Alternatively, any of the constructs is formulated with a poly(orthoester), including an auto-catalyzed poly(ortho ester) with any of avariable percentage of lactic acid in the polymeric backbone, andoptionally one or more additional excipients.Poly(D,L-lactide-co-glycolide)polymer (PLGA polymer) may be employed,preferably a PLGA polymer with a hydrophilic end group.

EXAMPLE 16

A patient with congestive heart failure, such as acutely decomponensatedcongestive heart failure with dyspnea at rest or with minimal activity,is administered a formulation including one or more of any of constructs1-1 to 1-248, 2-1 to 2-21, 5-1 to 5-18 and 6-1 to 6-12, including aformulation such as by any method of Example 10, by means ofsubcutaneous injection.

EXAMPLE 17

A patient with chronic congestive heart failure is administered a timerelease injectable formulation of Example 15 by means of an injection,such as a deep intramuscular injection, for example, in the gluteal ordeltoid muscle.

EXAMPLE 18

Applicants have also discovered an inverse correlation between theextent of resistance to digestion by neutral endopeptidase of aconstruct according to the invention and its pharmacokinetic clearance(CL), as shown in Table 7 below. Neutral endopeptidase (“NEP”) is anendogenous enzyme that inactivates and clears all three humannatriuretic peptides. NEP is present within renal tubular cells andvascular cells. NEP is highly homologous between mammalian species. Thepercent identity of NEP between Rat and Mouse is 98.5%, between Humanand Mouse is 93.6%, and between Human and Rat is 93.7%.

NEP resistance of various constructs of the invention was evaluatedusing the following experimental procedure. All constructs were dilutedin 0.1 M Tris-HCL buffer (pH 7.4) to 100 μM. 40 μL of diluted constructswere added to each tube and all tubes were kept on ice. 40 μL of dilutedNEP, either human recombinant NEP (R&D Systems, Minneapolis, Minn.,catalogue #1182-ZN) at 8 ng/μL or mouse NEP (R&D Systems, catalog#1126-ZN) at 2.5 ng/μL, was added to each tube. The tubes were mixedgently and spun. All tubes were then incubated at 37° C. for 0, 0.5, 1,1.5 and 2 hours. At the end of the incubation time, the reaction wasstopped by adding 5 μL of 10% TFA. Constructs were linearized using TCEP(Tris[2-carboxyethyl]phosphine. The extent to which NEP was activeagainst the constructs was then analyzed by HPLC and/or LC/MS. Dataanalysis included determining the percent of remaining startingmaterial, i.e., undigested peptide construct, and the percent andsequence of each proteolytic fragment.

TABLE 7 IV Dosing, PK cGMP hNEP Cl AUC AUCnom Cmax % of StartingMaterial Compound ID (mL/min/kg) (nM · hr) (nM · hr) (nM) 1 h 2 h 1-18(SEQ ID NO: 20) 55 102 119 172 91 82 1-43 14 238 119 242 97 92 1-44 1518 9 35 93 86 1-53 40 66 33 105 87 78 1-54 (SEQ ID NO: 38) 66 128 64 22085 82 1-55 (SEQ ID NO: 39) 21 116 144 170 83 70 1-63 (SEQ ID NO: 20) 20389 189 197 67 44 1-100 (SEQ ID NO: 29) 456 8 4 30 6 0 1-118 10 11 6 2796 92 1-130 92 276 138 524 75 56 1-131 21 161 81 422 87 70 1-133 12 169563 171 96 93

Accordingly, one embodiment of the invention provides a constructaccording to any of the formulas of the invention that demonstrates atleast 80% of starting material remaining after 1 hour under the hNEPresistance assay conditions described. A related embodiment of theinvention provides a construct according to any of the formulas of theinvention that demonstrates at least 90% of starting material remainingafter 1 hour under the hNEP resistance assay conditions described. Afurther related embodiment of the invention provides a constructaccording to any of the formulas of the invention that demonstrates atleast 95% of starting material remaining after 1 hour under the hNEPresistance assay conditions described. In a variation of any of theseembodiments, the construct also demonstrates at least 80% of startingmaterial remaining after 2 hours under the assay conditions. In arelated variation, the construct also demonstrates at least 90% ofstarting material remaining after 2 hours under the assay conditions.

Another embodiment of the invention provides a construct according toany of the formulas of the invention that demonstrates no more than 90%of starting material remaining after 1 hour under the hNEP resistanceassay conditions described. A related embodiment of the inventionprovides a construct according to any of the formulas of the inventionthat demonstrates no more than 80% of starting material remaining after1 hour under the hNEP resistance assay conditions described. In avariation of either embodiment, the construct also demonstrates no morethan 90% of starting material remaining after 2 hours under the assayconditions. In a related variation, the construct also demonstrates nomore than 80% of starting material remaining after 2 hours under theassay conditions.

Thus, constructs of the invention may be selected for pharmokineticproperties based on the extent of NEP resistance they demonstrate.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

Although the invention has been described in detail with particularreference to these preferred embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverall such modifications and equivalents. The entire disclosures of allreferences, applications, patents, and publications cited above and/orin the attachments, and of the corresponding application(s), are herebyincorporated by reference.

1. A cyclic peptide construct with an N-terminus and a C-terminuscomprising from eleven to thirteen amino acid residues with an aminoacid surrogate at the C-terminus position of the following formula:wherein

Y is CH₂ or C═O; W is CH₂, NH or NR″; Z is H or CH₃; J is —H, —OH,—C(═O)—OH, —C(═O)—NH₂, or a C-terminus capping group; one of R^(a) andR^(b) is H and the other of R^(a) and R^(b) is —(CH₂)_(y)—R″; R″ is:—NH₂, —NH—C(═NH)—NH₂, —NH—(CH₂)_(y)—NH₂, —NH—C(═O)—NH₂, —C(═O)—NH₂,—C(═O)—NH—CH₃, —C(═O)—NH—(CH₂)_(y)—NH₂, —NH—C(═NH)—NH-Me,—NH—C(═NH)—NH-Et, —NH—C(═NH)—NH—Pr, —NH—C(═NH)—NH—Pr-i, —NH—C(═O)—CH₃,—NH—C(═O)—CH₂—CH₃, —NH—C(═O)—CH—(CH₃)₂, —NH—C(═O)—O—CH₃,—NH—C(═O)—O—CH₂—CH₃, —NH—C(═O)—O—C—(CH₃)₃, —NH—C(═O)—NH—CH₃,—NH—C(═N—C(═O)—O—C—(CH₃)₃)—NH—C(═O)—O—C—(CH₃)₃,—N(C(═O)—O—C—(CH₃)₃)—C(═NH)—NH—C(═O)—O—C—(CH₃)₃,

R′″ is an acyl, a C₁ to C₁₇ linear or branched alkyl chain, a C₂ to C₁₉linear or branched alkyl acyl chain, a C_(i) to C₁₇ linear or branchedomega amino aliphatic, or a C_(i) to C₁₇ linear or branched omega aminoaliphatic acyl; x is 1 or 2; y is 1 to 5; n is 0, 1 or 2; and the carbonatoms marked with an asterisk can have any stereochemical configuration;with the amino acid surrogate at the C-terminus position beingcovalently bonded to the immediately adjacent amino acid residue.
 2. Thecyclic peptide construct of claim 1, wherein the C-terminus cappinggroup is: —(CH₂)_(m)—OH, —C(═O)—(CH₂)_(m)—N(v₁)(v₂),—C(═O)—O—(CH₂)_(m)—CH₃, —O—(CH₂)_(m)—CH₃, —O—(CH₂)_(m)—N(v₁)(v₂),—O—(CH₂)_(m)—OH, —C(═O)—NH—(CH₂)_(m)—S(v₁), —C(═O)—NH—(CH₂)_(m)—CH₃,—C(═O)—NH—(CH₂)_(m)—N(v₁)(v₂), —C(═O)—N—((CH₂)_(m)—N(v₁)(v₂))₂,—C(═O)—NH—CH(—C(═O)—OH)—(CH₂)_(m)—N(v₁)(v₂),—C(═O)—NH—(CH₂)_(m)—NH—C(═O)—CH(N(v₁)(v₂))((CH₂)_(m)—N(v₁)(v₂)), or—C(═O)—NH—CH(—C(═O)—N(v₁)(v₂))—(CH₂)_(m)—N(v₁)(v₂), including all (R) or(S) configurations of the foregoing; v₁ and v₂ are each independently Hor a C₁ to C₁₇ linear or branched alkyl chain; and m is in each instanceindependently 0 to
 17. 3. The cyclic peptide construct of claim 1,comprising at least one further amino acid surrogate at any positionother than the C-terminus position of the following formula:

wherein Q is a bond unless the surrogate is at the N-terminus positionof the construct, in which case Q is —H or an amine capping group; andR^(c) and R^(d) are each independently H or a natural or unnatural aminoacid side chain moiety or derivative of an amino acid side chain moiety.4. The cyclic peptide construct of claim 3, wherein the at least onefurther amino acid surrogate is at the N-terminus position of theconstruct and Q is an amine capping group of the formula:—(CH₂)_(m)—N(v₃)(v₄), —(CH₂)_(m)—CH₃, —(CH₂)_(m)—O(v₃),—(CH₂)_(m)—C(═O)-(v₃), —(CH₂)_(m)—C(═O)—O-(v₃), —(CH₂)_(m)—S(v₃),—C(═O)—(CH₂)_(m)—CH₃, —C(═O)—(CH₂)_(m)—N(v₃)(v₄),—C(═O)—(CH₂)_(m)—C(═O)-(v₃), —C(═O)—(CH₂)_(m)—O(v₃), or—C(═O)—(CH₂)_(m)—S(v₃); v₃ and v₄ are each independently H, a C₁ to C₁₇linear or branched alkyl chain or a C₂ to C₁₉ linear or branched alkylacyl chain, on the proviso that if one of v₃ or v₄ is an alkyl acylchain, then the other of v₃ or v₄ is H; and m is 0 to
 17. 5. The cyclicpeptide construct of claim 1, further comprising an N-terminus acylcomprising a C₂ to C₁₈ linear alkyl, a C₃ to C₁₇ branched alkyl, a C₂ toC₁₈ linear alkenyl or alkynyl or a C₃ to C₁₈ branched alkenyl oralkynyl.
 6. The cyclic peptide construct of claim 5 wherein theN-terminus acyl is −C(═O)—(CH₂)_(p)—CH₃ where p is 1 to
 18. 7. Thecyclic peptide construct of claim 6 of the formula:


8. The cyclic peptide construct of claim 1, wherein the amino acidresidues comprise natural or unnatural α-amino acids, β-amino acids,N-substituted amino acids, or α, α-disubstituted amino acids, includingall (R) or (S) configurations of any of the foregoing, or anycombination of the foregoing.
 9. The cyclic peptide construct of claim1, wherein at least two of the amino acid residues are joined by anon-peptide bond.
 10. The cyclic peptide construct of claim 9, whereinthe at least one non-peptide bond is —CH₂—NH—, —CH₂—S—, —CH₂—O—, or—C(═O)—CH₂—, an isostere of any of the foregoing, or —CH₂—CH₂— or—CH═CH—.
 11. The cyclic peptide construct of claim 1, wherein the cyclicpeptide construct binds to a natriuretic peptide receptor selected fromthe group consisting of a receptor for ANP, BNP, CNP, sCP, DNP, TNP-a,TNP-b or TNP-c.
 12. The cyclic peptide construct of claim 11, whereinthe cyclic peptide construct exhibits, upon administration to a mammal,one or more advantages relative to the corresponding amino acid sequencenot comprising an amino acid surrogate, the advantages selected from thegroup consisting of increased resistance to enzymatic degradation,increased circulation half life, increased bioavailability, increasedefficacy, and prolonged duration of effect.
 13. The cyclic peptideconstruct of claim 1, wherein Y is C═O, W is NH, Z is H, x is 1 and n is0.
 14. The cyclic peptide construct of claim 1, further comprising atleast one prosthetic group covalently bonded to a reactive group in aside chain or terminal group of at least one of the amino acid residuesor to a carboxyl group, amine group or reactive group in a C-terminuscapping group of the amino acid surrogate.
 15. The cyclic peptideconstruct of claim 14, wherein the prosthetic group comprises at leastone polymeric sequence comprising repeat units including carbon andhydrogen atoms.
 16. The cyclic peptide construct of claim 15, whereinthe polymeric sequence is poly(alkylene oxide), poly(vinyl pyrrolidone),poly(vinyl alcohol), polyoxazoline or poly(acryloylmorpholine).
 17. Thecyclic peptide construct of claim 16, wherein the poly(alkylene oxide)is poly(ethylene glycol) (PEG).
 18. The cyclic peptide construct ofclaim 17, wherein the prosthetic group comprising PEG is derivatizedwith a linking group.
 19. A pharmaceutical composition, comprising acyclic peptide construct of claim 1 or a pharmaceutically acceptablesalt of a cyclic peptide construct of claim 1 and a pharmaceuticallyacceptable carrier.
 20. A pharmaceutical composition, comprising acyclic peptide construct of claim 7 or a pharmaceutically acceptablesalt of a cyclic peptide construct of claim 7 and a pharmaceuticallyacceptable carrier.